TS. CHU HONG MU

C S V PHNG PHP SINH HC PHN T

NH XUT BN I HC S PHM

Li ni u.................................................................................................................................4

Chng 1: H GENE..............................................................................................................5 1. KHI NIM H GENE (GENOME) ..................................................................5 2. AXIT NUCLEIC ..................................................................................................9 3. ADN V TI BN ADN..................................................................................11 4. ARN V C CH PHIN M .........................................................................19 THO LUN...........................................................................................................23

Chng 2: C IM CU TRC GENE CA SINH VT PROKARYOT V EUKARYOT ...........................................................................................................25 1. C IM CU TRC GENE PROKARYOT ..........................................25 2. CU TRC PHN ON GENE EUKARYOT .........................................25 3. MT S TRNH T ADN................................................................................27 THO LUN...........................................................................................................32

Chng 3: MI LIN H GIA ADN, ARN, PROTEIN................................................33 1. THNG TIN DI TRUYN V MT M DI TRUYN .................................33 2. PROTEIN ...........................................................................................................39 3. QU TRNH SINH TNG HP PROTEIN.....................................................42 4. IU HO BIU HIN GENE ........................................................................45

THO LUN......................................................................................................................49

Chng 4: ENZYME S DNG TRONG K THUT SINH HC PHN T ............50 1. ENYME GII HN (RESTRICTION ENZYME HOC ENZYME CT HN

CH (RESTRICTION ENDONUCLEASE - RE) .............................................50 2. CC NHM ENZYME KHC.........................................................................57 3. ENZYME NUCLEASE......................................................................................60

THO LUN......................................................................................................................61

Chng 5: XC NH MI LIN QUAN GIA PROTEIN V C TNH SINH VT ...............................................................................................................62 1. MT S LOI PROTEIN CHC NNG........................................................62 2. QUY TRNH XC NH CH TH PHN T PROTEIN..............................66 3. K THUT PHN TCH PROTEIN .................................................................67 4. K THUT PHN TCH ENZYME..................................................................74 5. NGHIN CU RIPS BNG WESTERN BLOT ..............................................77

THO LUN......................................................................................................................78

Chng 6: K THUT SINH HC PHN T TRONG PHN TCH H GENE SINH VT..........................................................................................................................79 1. PHNG PHP TCH CHIT ADN..............................................................79 2. LAI PHN T ...................................................................................................83 3. XC NH TRNH T NUCLEOTIT CA ADN ..........................................85 4. RFLP TECHNOLOGY (K thut phn tch hin tng a hnh ca di cc

phn on ADN).................................................................................................87

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THO LUN......................................................................................................................91

Chng 7: PHN NG CHUI POLIMERASE (POLIMERASE CHAIN REACTION - PCR)................................................................................................92 1. PHN NG CHUI POLIMERASE (PCR) ....................................................92 2. PHN TCH TNH A HNH CA ADN C NHN BN NGU

NHIN (RANDQM AMPLIFIED PQLIMORPHIC DAN - RAPD) ................99 4. PHN TCH TNH A HNH CHIU DI CC PHN ON ADN C

NHN BN C CHN LC .........................................................................106 THO LUN.........................................................................................................108

Chng 8: T BO CH V VECTOR ..........................................................................109 1. T BO CH ..................................................................................................109 2. VECTOR ..........................................................................................................111 3. PHNG PHP CHUYN VECTOR TI T HP VO T BO CH ..122 THO LUN.........................................................................................................125

Chng 9: PHN LP GENE TCH DNG PHN T V BIU HIN GENE......126 1. PHN LP GENE ...........................................................................................126 2. TCH DNG GENE .......................................................................................127 3. BIU HIN GENE...........................................................................................139 THO LUN.........................................................................................................151 Ti liu tham kho chnh .....................................................................................152 nhng cng trnh ca tc gi cng cc cng tc vin cng b.....................155

C S V PHNG PHP SINH HC PHN T .....................................................162

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Li ni u Sinh hc phn t hin i ang pht trin mnh v tr thnh nng ct ca Cng ngh sinh hc. Vit Nam, thnh tu nghin cu Sinh hc phn t v p dng k thut phn t trong nghin cu Khoa hc s sngv Cng ngh sinh hc c nhiu ng gg trong vic chm sc, bo v sc kho con ngi; trong nh gi ti nguyn sinh vt; trong chn ging v sn xut nng lm ng nghip. Sinh hc phn t l mn Sinh hc hin i, c ging dy nhiu trng i hc v ang c nhng ng gp nht nh trong o to lp ngi c tri thc v Cng ngh sinh hc gp phn vo s nghip Cng nghip ho, hin i ho C s v phng php Sinh hc phn t c bin son t nhiu ti liu, bi ging v cng trnh nghim cu mi v Sinh hc phn t hin i ca cc tc gi trong v ngoi nc, nhm cung cp nhng kin thc c bn v nguyn l v ng dng ca Sinh hc phn t, lm ti liu cho nghin cu, ging dy v hc tp mn ny trng i hc. Cun C s v phng php Sinh hc phn t c cu trc bi 9 chng: Chng 1. H gene

Chng 2. c im cu trc gene ca sinh vt Prokaryot v Eukryot Chng 3. Mi lin h gia ADN, ARN, Protein

Chng 4. Enzyme s dng trong k thut sinh hc phn t Chng 5. Xc nh mi lin quan gia protein v c tnh sinh vt Chng 6. K thut sinh hc phn t trong tch h gene sinh vt Chng 7. Phn ng chui polimerase (PCR) Chng 8. T bo ch v Vector Chng 9. Phn lp gene, tch dng phn t v biu hin gene.

Tc gi trn trng cm n PGS.TS. Nguyn Trng Lng, TS. Lng Th Hng Vn c v gp cho bn tho, xin cm n nhng ng gp ca Hi ng nghim thu nh gi gio trnh ca trng i hc S phm - i hc Thi Nguyn v cm n cc kin ng gp qu bu ca ng o cc nh khoa hc. Trong qu trnh bin son chc chn c nhng sai st, tc gi rt mong nhn c nhng gp ca bn c. Mi ng gp xin gi v Khoa Sinh - K thut Nng nghip trng i hc S phm - i hc Thi Nguyn.

Tc gi

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Chng 1: H GENE Tm tt: S ra i ca sinh hc phn t c nh du bng thi im m Oatsn v Cric (1953) pht hin ra cu trc ADN. Tri qua hn 40 nm (1953 - 2000) sinh hc phn t t c nhng thnh tu v i m nh cao ca s pht trin ny l nhng khm ph bn cht sinh hc ca s sng cp phn t v xy dng cc k thut sinh hc phn t ng dng vo thc tin. Geneomics v Proteomics l vn ang c c bit quan tm hin nay m c s ca cc lnh vc ny l nhng pht hin v cu trc v chc nng ca axit nucleic, v c im ca genome nhn, genome ti th, genome lp th. Nhng im khc nhau v cu trc v chc nng ca cc h gene cho php ng dng vo thc t chn ging v nghin cu ngi. Cng vi cu trc ADN v ARN cn c c im ca qu trnh ti bn ADN v phin m cng c quan tm, v n l c s ca nhng k thut sinh hc phn t - cc thao tc ADN v ARN. Ni dung c bn ca chng cp n nhng c s ca sinh hc phn t. Ni dung ca chng gm 4 vn c bn: (1). Khi nim h gene; (2). Axit nucleic; (3). ADN v ti bn ADN, (4). ARN v c ch phn m.

1. KHI NIM H GENE (GENOME) Qu trnh sinh trng v pht trin ca sinh vt tri qua rt nhiu giai on v tt c qu trnh u ph thuc vo s iu khin ca cc gene. Cu trc, chc nng ca t bo c quyt nh trc tip bi protein, l sn phm cui cng ca s biu hin gene. Qu trnh th hin hot ng ca gene qua protein, b nh hng rt ln bi cc yu t ngoi cnh nh nh sng, ch dinh dng, s cng sinh v s tng tc ca cc gene trong h gene ca t bo. Nh vy, gia cc thnh phn ca h gene trong t bo sng c s tng tc vi nhau v c mi quan h vi cc yu t ca mi trng. Trong t bo, bn cnh genome trong nhn, h thng di truyn cn phn b trong lc lp, ti th v plasmid (vi khun). Cc c quan t ny cng tham gia vo qu trnh sinh tng hp protein ca t bo. T bo l n v cu trc v chc nng c bn ca cc c th sng, t bo cn l n v ca s di truyn. H gene l ton h cc gene trong t bo ca c th sinh vt, h gene ha ton b thng tin di truyn c trng cho tng loi, cho tng c th trong loi. sinh vt Prokaryot h gene gm ton b cc gene trong t bo; cn Eukaryot

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h gene gm ton b cc gene trong t bo n bi (n). T bo n bi c mt h gene, cc sinh vt hoc t bo lng bi c hai h gene, sinh vt a bi c nhiu h gene. H gene ca sinh vt Eukaryot bao gm h gene nhn (genome nhn), h gene ti th (genome ti th), h gene lc lp (genome lc lp). H gene Prokaryot ch c mt thnh phn gm cc on ADN khng ging nhau; cn h gene Eukaryot c ba thnh phn ADN khng ging nhau: Cc on ADN lp li nhiu (chim khong 25%), cc on ADN lp li t (30%) v cc on ADN khng lp li (45%). 1.1. H gene nhn H gene nhn l genome ln nht trong t bo xt v mt khi lng cng nh s lng gene m ho. ADN nhn c sp xp gn trong nhim sc th trong s lin kt vi protein cha histone v khng cha histone. ADN c vai tr m ho thng tin di truyn, cn protein th bo v v tham gia iu khin s sao chp, phin m c chnh xc. Qu trnh c bn trong pht trin ng, thc vt l s nhn i vt cht di truyn v phn u cho cc t bo con khi t bo phn chia. T bo thc vt cha mt lng ln ADN, khi lng ny thay i theo loi. Arabidosis thaliana c lng ADN nh nht (0.07 picogram). Allium cepa c lng ADN nhiu hn 33,5 pg/ haploid genome (h gene n bi). 1.2. H gene lc lp Cho n nm 1962, ngi ta mi khm ph ra ADN v ribosom c trong lc lp. Nh vy, lc lp hoc trong lp th cha tt c b my cn thit t mnh biu hin hot ng gene. H gene lc lp l ton b cc gene trong lc lp hay ton b lng thng tin di truyn cha trong ADN ca lc lp. Lng ADN trong lc lp rt ln, chim ti 15% tng lng ADN c th thc vt, ribosom chim 60% lng ribosom ca t bo, ADN lc lp c cu to bi cc phn t c cu trc mch vng, c trng lng phn t 83 - 128 x 106, trong gn 85% phn t ADN l mch n. ADN lc lp lm khun phin m tng hp mARN chloroplast. y cc phn t mARN ch c khong 20 nucleotit, chng thc hin s tng hp protein ti ch trong chloroplast. Ribosom trong chloroplast l 70S bao gm 30S v 50S. Sau khi tng hp xong protein c vn chuyn n ni m lc lp cn thit. V c bn h thng di truyn trong lc lp tng t vi h thng di truyn ca sinh vt Prokaryot. Do vy c nhiu cuc tranh lun, c phi vt liu di truyn ca sinh vt tin nhn l ngun gc ca ADN lc lp? Ngoi ra ADN ca lc lp khng cha mt s gene c trng cho nhng sinh vt c nhn tht. iu ny tm thy trong ADN thuc l v ng. Ribosom ca lc lp cng ging

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ribosom ca sinh vt Procariot: 70S c cu to bi 2 tiu phn 50S v 30S. Ribosom ca E. coli v lc lp thc vt c c tnh min dch ging nhau. Thng qua vic nghin cu sn phm biu hin gene l protein, ngi ta pht hin gia h gene lc lp v h gene nhn trong t bo thc vt c mi quan h cht ch. - Genome ca lc lp khng c kh nng tng hp tt c cc protein c trong chng. - Phn ln polipeptid ca lc lp c tng hp do genome trong nhn t bo. - Nhng polipeptid ny c chuyn vo lc lp theo c ch sau dch m thc hin qua v lc lp. - H thng di truyn lc lp iu khin tng hp cc protein cn cho s pht trin ca chng vi s lng khong 100 polipeptid.

Bng 1.1. Kch thc mt s ADN lc lp (ct. ADN), ADN nhn v vi khun

AND Kch thc (bp) Kch thc vng ( m)Plasmid 1 200 x 103 - Trc khun E-coli (chromosome) 3,8 x 106 - Trng roi (Euglena) (ct. ADN) 1,4 x 105 44 - 44 Thuc l (Tabaco) (ct. ADN) 1,6 x 105 - Ng (Maize) (ct. ADN) 1,36 x 105 43 u xanh (mungbean) (ct. ADN) 1,21 x 105 39 - Polipeptid c m ho tng hp v ARN trong lc lp m nhim chc nng ca c quan t lin quan n qu trnh quang hp. la ngi ta xc nh c v tr ca cc gene quan trng iu khin qu trnh quang hp nh rbcl, atpBE, pSbA, pstA, psbA. Cc gene ny ng vai tr quan trng trong qu trnh sinh tng hp protein h thng quang ho II. 1.3. H gene ti th (Mitochondria Genome)

nghin cu h gene ti th iu quan trng u tin l thu c ADN ti th. C th ly v d cy la theo Salech v cng s (1989), phng php phn tch ADN trong ti th gm cc bc sau: - Li tm vi tc chm loi b nhn, lc lp v cc thnh phn khc ca t bo. - Li tm vi tc nhanh tch ti th. - Li tm vi tc chm ly nhn, lc lp v cc thnh phn khc.

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- X l ADN- ase I phn gii cc cht ADN nhn dnh trong ti th. - Lm sch ti th. - Tch chit mtADN t ti th (mtADN). Nh vy ngi ta c th thu c ADN ca mitochondria tinh sch phc v cho cc cng tc nghin cu tip theo. mtADN th hin mt s khc bit rt ln v mt kch thc v hnh dng. i vi ng vt, mtADN c dng vng v kch thc xp x 15 - 20kb. Ngc li mtADN ca thc vt c nhiu dng (vng.,thng va(cc dng khc), cng nh c kch thc ln hn nhiu: cy bp ci l 200kb, hoc mt loi da l 2500 kb. Do vy genome ti th thc vt tng i ln v gm mt vi phn t ADN. Lng mtADN trong thc vt chim khong 0,5 - 1% lng ADN ca t bo nhng n ng vai tr sng cn cho s pht trin v sinh sn thc vt. Chc nng ca mtADN ging nh ctADN. N c kh nng m hoatng hp mt s lng nh pohlieptid nhng rt quan trng.,phc v cho nhng hot ng ca chnh n. Nhng sn phm chnh m mtADN iu khin tng hp l 3 tiu phn ca cytochromoxydaza, 2 tiu phn ca phc cht cytochrom bc v mt s thnh phn khc (Andre, 1991). Ngoi ra mtADN cn ng vai tr quan trng trong hin tng bt dc c t bo cht (CMS). y l mt c tnh c khai thc trong sn xut cc dng lai nhiu cy trng nh la, ng... Spruill v cng s (1981) ch ra cy ng c s khc nhau gia mtADN trong t bo cy thng v trong t bo cy c hin tng CMS. Khi phn tch sn phm protein th nhng mtADN cy CMS c kh nng tng hp chui peptid 130000 MW, nhng n mt kh nng tng hp chui polipeptid 21000 MW nguyn sinh cht t bo cy bnh thng. Mt khc ngi ta cn thy hin tng bt dc t bo cht cn lin quan n nhng phn t ADN ging nh plasmid tm thy trong ti th. Chng hn ng trong t bo cht ca cy CMS c mang hai phn t AND mch thng vi kch thc 6,2 kb v 5,2 kb. Vic s dng code m ha tng hp protein ca mtADN c nhng im cn lu : trong h thng hot ng m di truyn thng thng code UGA cung cp tn hiu kt thc qu trnh tng hp mt chui polipeptid, nhng ADN ca ti th n li l code ca tryptophan. Ngc li ADN nhn code CGG m ho cho tryptophan th ti th n li m ho arginin. Ngoi ra ngi ta pht hin mt dng mi ca mARN trong genome ti th cy trng v n c gi l ARN - Editing. y l mt vn th hin s tin ho ca thc vt bc cao, gip cho n c kh nng

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thch nghi hn vi iu kin ngoi cnh.

2. AXIT NUCLEIC 2.1. Axit nucleic l vt cht di truyn Axit nucleic bao gm axit deoxyribonucleie (ADN) hoc axit ribonucleic (ARN) u p ng tiu chun ca vt cht di truyn. C rt nhiu bng chng chng t ra axit nucleic l mt vt cht di truyn cp phn t. Axit nucleic hp th tia t ngoi cc i bc sng 260nm v iu ny ph hp mt cch chnh xc vi bc sng m tia t ngoi c th gy t bin ti a t bo, trong khi protein bc sng 280nm. - Nm 1928 F. Grifflth pht hin thy ni ph cu khun S, khun lc nhn do c v bc polisaccharid (vi khun gy vim phi) ca vi khun Diplococus pneumoniae lm chut cht khi tim vo c th chut. Trong khi khun lc R (khun lc khng c v bc polisaccharid) khng gy c hi g. Hn hp cc ph cu khun R cn sng v ph cu khun S cht th lm cho chut b vim phi, cht v t mu ca chng phn lp c vi khun S sng. Nh vy tc nhn no t ph cu khun bin np v lm cho ph cu khun R thnh S. Ngi ta chng minh c rng: ADN tch t vi khun S nu c a vo vi khun R th gy nn s bin i kiu R S, pht hin ny c khng nh ADN l vt cht di truyn. Nm 1944 O. T Avery, Mc Leod v Mc Carti chng minh c rng tc nhn bin np l ADN. Ph cu khun S b x l bng protease hoc ARN-aza th hot tnh bin np vn cn: nhng nu ph cu khun S b x l bi ADN-aza th hot tnh bin np khng cn na.

Hnh 1.1. S chng minh vt cht di truyn l ARN

Nm 1957, H. Fraen Kel - Conrat v B. Singer cng b th nghim virut m thuc l c li ARN v v protein, c hai dng a v b. Cc th nghim lp rp li ARN ca dng ny vi protein ca dng kia v ngc li thnh cng to ra virut c v v li thuc hai dng khc nhau. Sau em gy nhim vo

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thuc l, kt qu cc virut con phn lp c t vt m u mang c v protein v li ARN thuc cng mt dng l dng ca li ARN mang nhim ch khng phi ca v protein. Nh vy thng tin di truyn c cha ng trong ARN ch khng phi trong protein. 2.2. Axit nucleic virut, Prokaryot v Eukaryot Virut l vt cht sng c cu to rt n gin, gm li l axit nucleic v v l protein. Vt cht di truyn ca virut c hai loi: ADN v ARN. a s cc loi virut u sng k sinh, khi xm nhp vo t bo vt ch (c th ton b virut hay ch A. nucleic). Virut sng k sinh t bo Prokaryot c gi l phage hay bacteriophage (thc khun th). Mt s virut c li l ARN ch yu k sinh thc vt, cn cc virut k sinh ng vt v t bo Prokarvot a s c li l ADN. NST phi to ra mi trng vt l cho phn t ADN hot ng, tc ng qua li vi cc h thng trao i cht trong t bo. H thng sp xp cch t chc, b tr ADN trong NST nhng t chc sinh vt cha c nhn chnh thc v sinh vt nhn chun c s khc nhau nh th no? Ngi ta s dng ba hng chnh nghin cu t chc ADN trong cc i tng sinh vt khc nhau, l vic s dng cc thit b v k thut nh knh hin vi in t, nhiu x tia X, x l bi enzyme bin i cu trc NST. i vi sinh vt Prokaryot, t chc ADN trong NST dng nucleoid (vng nhn). Vng nhn cha ADN v ADN c gp v cun thnh nhiu vng xon: ADN dng siu xon, tnh cht siu xon chu s kim sot ca enzyme topoisomerase. V d E. coli ADN c kch thc 300 m khi co ngn kch thc di 25 m v tip tc co ngn th kch thc ch cn 1,5 m. Cch sp xp ny c pht hin nh enzyme ribonuclease v deoxyribonuclease. Prokaryot cha ADN trn, chui kp, dng vng, ngoi ra vt cht di truyn cn plasmid (chim l-2%). E. coli ch c 1 phn t ADN dng vng cha 3000-4000 gene. i vi sinh vt Eukaryot, ADN trong t bo c c trong nhn, ti th v lp th v ch yu ADN nm trn nhim sc th trong nhn t bo. ADN nhn c dng mch kp, ch yu l B-ADN. ADN ti th v ADN lc lp c dng kp vng. T chc ADN trong nhim sc th dng nucleosome (th nhn). Mi nucleosome gm phn t ADN v protein (kim) c gi histon. Histon gm 8 phn t (octamer). Phn bit 5 loi histon vi t l gn nh nhau H1, H2A, H2B, H3, H4. Trong c 2 phn t H3, 2 phn t H4 lin kt vi vng trung tm; 2 phn t H2A, 2 phn t H2B lin kt vng ngoi.

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Cc nucleosome ni vi nhau bng mt on nucleotit (ADN) di 15 - 100 cp nucleotit. NST c cu trc xon theo nhiu bc: tp hp nhiu nucleosome thnh si c bn c cu trc xon (100), si c bn tip tc xon thnh solenoit (si nhim sc) c ng knh l: 250: solenoit cun xon ln na to nn ng rng c ng knh 2000; ng rng ny cun xon mt ln na thnh si chromatit c ng knh bng 6000.

3. ADN V TI BN ADN 3.1. c im cu trc ca phn t ADN Hm lng ADN trong nhn t bo c trng cho tng loi v ph thuc v s lng nhim sc th trong nhn. ADN l i phn t, c khi lng v chiu di rt ln. sinh vt Prokaryot mi nhim sc th c thc nhiu phn t ADN, cn sinh vt Eukaryt mi nhim sc th ch c mt phn t ADN.

Bng 1.2. Hm lng ADN ca mt s loi sinh vt Ngi Rui gim

T bo lng bi (2n) 6,6 pg ADN 2 pg ADN

T bo n bi (n) 3,3 pg ADN. 1 pg ADN.

1 picogram (pg) ADN = 0,965. 109 bp = 6,1.1011 dalton = 29cm Ngi ta phn bit cc dng ADN l B, A. Z, C, D. Cc dng AND khc nhau bi chiu xon, chiu cao mt chu k xon, s cp nucleotit trong mt chu k.

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Bng 1.3. So snh c im ca mt s loi AND

Dng AND

Chiu xon

S cp nucleotit

chu k

Gc xon (0)

ng knh ()

Chiu cao mt chu k

xon

Dng thit in

B Phi 10 36 20 34 Trn A Phi 11 32,7 23 28 Trn Z Tri 12 30 18 37,1 zigzac C Phi 9,3 38,6 20 31 Trn D Phi 8 - - - Bt gic

Oatsn v Cric (1953) xy dng m hnh B-ADN (ph bin nht), l chui xon kp gm 2 mch polinucleotit xon theo chiu t tri sang phi. M hnh ca Oatsn v Crick l gii c nhng chc nng c bn ca ADN l bo m cho vic ti sinh trong qu trnh t sao chp gian k v iu chnh vic tng hp cc enzyme v cc protein. y l mc thi gian nh du s ra i ca ngnh sinh hc phn t. ADN v ARN u l nhng polime gm nhiu monomer ni vi nhau, mi monomer gi l nucleotit ( ADN gi l deoxynucleotit, cn ARN l ribonucleotit). Mi deoxynucleotit v ribonucleotit gm 3 thnh phn baz nit ( purin v pirimidin) ng pentose v axit photphoric.

Hnh 1.2. S cu to phn t ng deoxyribose v ribose

Cc baz purin (kch thc khong 7) gm Adeine (A); Guanine (G); cn cc baz pirimidin (kch thc khong 5) gm Thymine (T); Cytosine (C); Uracil (U).

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Hnh 1.3. S cu trc ca cc loi baz nit

Hnh 1.4. S cu to cc deoxynucleotit

Kt qu nghin cu ca Chargaff v phn tch hm lng baz purin v pirimidin trong ADN thy rng t l Adenine bng Thymine v t l Guanine bng Cytosine (A = T; G = C). ADN gm hai chui polinucleotil, mi mch polinucleotit c nhiu nucleotit lin kt vi nhau bng mi lin kt phosphodieste. Cc baz i din trn hai mch n lin kt vi nhau bng mi lin kt hydro theo nguyn tc b sung (A - T; G - C). Hai mch polinucleotit ca ADN c chiu ngc nhau: 13

3'OH 5'P 5'P 3'OH

Tnh c trng ca phn t ADN ph thuc vo 3 yu t: s lng, thnh phn v trnh t sp xp cc nucleotit trong ADN, trong ph thuc nghim ngt vo trnh t cc nucleotit. Kch thc ca phn t ADN c tnh bng bp (base pair- cp baz) hoc kb (kilobase - kb = 1000 bp), v tnh c hiu ca nucleotit l do baz, cho nn khi ni n axit nucleic th baz thng c dng thay cho nucleotit.

Hnh 1.5. S chui polinucleotit ca B-ADN 3.2. Ti bn ADN 3.2.1. Cc kiu ti bn ADN Ngi ta a ra 3 gi thuyt v cc kiu ti bn ADN: Kiu bo ton: Chui ADN m gi nguyn, ADN mi c tng hp t nguyn liu (cha c bng chng trong t nhin). Kiu phn tn: ADN ban u t ra tng on nh, mi on nh lm khun tng hp cc on nh khc. Cc on nh ni li vi nhau thnh ADN. Kiu bn bo tn: Meselson v Stahl chng minh 1958 E. coli s dng ng v phng x N15. Mathew Meselson v Franklin Stahl (M - California) tin hnh th nghim nui E. Coli vi ngun N15, chng minh c c ch ti bn ADN. 3.2.2. H enzyme ti bn ADN 14

Ba loi enzyme ADN-polimerase E. Coli - ADN-poIimerase I: gi vai tr trong ti bn ADN, c th d sa cha. - ADN-poIimerase II: xc nh s bt u tng hp ADN. - ADN-poIimerase III: iu khin tng hp ADN, thc hin nhim v ch yu gia tng chiu di ca mt si mi.

Ba loi enzyme ADN - polimerase Eukaryot - ADN-polimerase (120.000 - 300.000 dalton) c chc nng ti bn ADN ca nhn.

- ADN-polimerase (30.000 - 50.000 dalton) sa i ADN.

- ADN-polimerase (150.000 - 300.000 dalton) ti bn h gene ti th. 3.2.3. C ch ti bn ADN theo Okazaki -Ti bn na gin on (Semidiscontinous replication ) 3.2.3.1. Hin tng dui xon

Hin tng trn c s tham gia ca mt loi protein l SSB (Single Strand Binding) bm vo si n ADN lun trng thi m xon, mi SSB bm vo 8 baz v mi chc ti bn c 250 SSB. 3.2.3.2. Khi u ti bn bng ARN mi (primer) ARN polimerase hot ng tng hp on ARN mi ngn to ra u 3'OH t do, sau ADN polimerase III hot ng ti bn. i vi E. coli primer c to ra bi enzyme primase. 3.2.3.3. Ko di, loi b mi v hnh thnh cc phn on Okazaki Sau khi ARN mi c tng hp, th ADN-polimerase I hot ng loi b mi nh ct t (5' - 3') v thay vo l mt on ADN mi (hnh thnh phn on Okazaki). Mi phn on Okazaki c 1000 - 2500 baz. Cc phn on Okazaki ni vi nhau bng ADN-ligase. Si ADN mi c tng hp bng cch ni cc phn on okazaki c gi l si ra chm (lagging strand) l si c tng hp khng lin tc (gin on); si kia c tng hp lin tc trn khun ca si ADN c chiu 3' - 5'. C ch ti bn ADN c mt si c tng hp lin tc v mt si c tng hp gin on c gi l ti bn na gin on. - Mch ADN mi c tng hp theo chiu 5' - 3', mt mch polinucleotit c tng hp lin tc v mt mch c tng hp gin on- Mi bc ca 15

qu trnh ti bn u c s tham gia ca cc loi enzyme tng ng. - Qu trnh lin kt vi cc nucleotit thc hin theo nguyn Ltc b sung (A - T, G - C ). - C s tham gia ca primer (ARN mi). - Mi ADN con c to thnh u cha mt mch c v mt mch mi (bn bo tn).

Hnh 1.6. Ti bn ADN theo Okazaki

3.2.4. Ti bn ADN ca virut - Cc phage (virut k sinh vi khun) mang ADN 1 si hoc 2 si, virut ARN c loi ARN 1 si v cng c th mang ARN 2 si. - C ch ti bn ADN 1 si virut x 174 (nghin cu nhiu nht) k sinh vi khun E. coli (hnh 1.7).

Hnh 1.7. Ti bn virut 174

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Ch c si (+) c ti bn v ch c 1 trong 2 si trong RF lm khun tng hp ra si (+). 3.2.5. Ti bn ADN t bo ca sinh vt Prokaryot 3.2.5.1. Ti bn ADN vng Prokaryot E. coli, qu trnh ti bn xut pht t mt im ori (origine) c gi l im xut pht v trin khai ra c hai pha. E. coli ch c mt im xut pht sao chp ori nn c ADN thnh mt n v sao chp (replicon). Prokaryot thng ch c mt replicon (hnh 1.8).

Hnh 1.8. n v ti bn (replicon) Prokaryot

3.2.5.2. Ti bn kiu ln ai thng (Rolling circle ) - E. coli F+ v F- tip hp. Yu t F c truyn t F+ sang F- v F- bin thnh F+, ADN vng plasmid c ti bn theo kiu ln ai thng to thnh ADN vng mi t bo nhn F-.

Hnh 1.9. Ti bn kiu ln ai thng (rolling circle)

3.2.6. Ti bn t bo ca sinh vt Eukaryot sinh vt Eukaryot s sinh sn ca t bo l mt qu trnh sinh trng phn nhn v phn bo mang tnh chu k, qu trnh c gi l chu k t bo (cell cycle). Chu k t bo gm 4 pha: Pha G1 (Gap 1) tip sau pha phn chia, nhim sc th tho xon tr v

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dng si mnh v xy ra qu trnh tng hp cc cht cho s nhn i ADN v din ra hng lot cc bin i dn n khi u cho s sao chp ADN. Pha S (Synthesis): vt cht di truyn ca cht nhim sc c nhn i, l qu trnh ti bn ADN, cc bng chng chng t rng sao chp ADN ca sinh vt nhn chun cng din ra theo kiu na gin on nh sinh vt nhn s. Cc enzyme cn thit cho s sao chp ADN ca sinh vt nhn chun bao gm nhm ADN-polimerase c ADN polimerase , ADN-polimerase v ADN-polimerase . Theo quan nim hin nay, cc sinh vt nhn chun bc cao ni chung - polimerase c s dng sao chp ADN ca nhn, -polimerase hot ng nh enzyme sa cha,

-polimerase chuyn trch sao chp ADN ti th.

Hnh 1.10. Cc s kin din ra mc phn t trong chu k t bo

M. Pha phn chia G1: Pha sau phn chia, chun b tng hp cc cht, chun b tng hp ADN S: Pha tng hp trong din ra qu trnh ti bn ADN G2: Pha trc khi phn chia t bo, trong din ra tng hp tubulin v cubuin 1. Bt u phn chia; 2. Kt thc phn chia;3. Xut hin nhn t cm ng tng hp ADN

sinh vt nhn chun, mi NST c nhiu n v sao chp (Replication Unit) cn gi l replicon, mi replicon c mt im khi u v hai im kt thc qu trnh sao chp. Cc NST ca sinh vt nhn chun bao gm ADN v histon cng nm trong th nhn. Nhng bng chng cho thy cc gene m ho histon sao m to ra cc bn sao tng hp s lng histon cn thit cho s hnh thnh cc th nhn mi trong pha S.

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Pha G2 (Gap 2): cc nhim sc th chun b cho nguyn phn. Mt s kin quan trng trong G2 l ni tng hp protein tubulin, cubulin c trng hp ho to ra cc vi ng ca b my thoi sc phn li cc NST trong nguyn phn. Pha M (Mitosis): Din ra qu trnh nguyn phn ca t bo, c im c bn ca nguyn phn l c nhng bin i ln v chc nng v cu trc ca t bo. Eukaryot c s lng ADN ln, ti bn ADN phc tp hn v tc chm (50 nucleotit/giy). Ti bn ca Eukaryot c nhiu replicon v nhiu im ori. 3.2.7. Sa sai trong ti bn Hng sao chp ADN thc hin theo chiu t 5'-3' v hng sa cha sai st trong ti bn li din ra theo chiu t 3'-5'. Enzyme ADN-polimerase I, III c hot tnh ca exonuclease (enzyme c hot tnh ct nucleotit u t do ca ADN), nu c nucleotit lp sai th ADN-polimerase s ct b nucleotit sai theo hng 3'-5'.

Hnh 1.11. Ti bn Eukaryot (nhiu replicon)

Trong trng hp ADN trng thi khng ti bn th c xp x 20 loi enzyme r sot dc cc NST tm ADN c bin i cu trc, 5 enzyme kim sot s sai st ca lin kt ho tr, mt s enzyme khc pht hin sai st bt cp gia cc baz khng b sung. Tng s c xp x 50 enzyme chuyn pht hin v sa cc sai st trong ADN.

4. ARN V C CH PHIN M 4.1. Cu trc v chc nng ca ARN 4.1.1. Cc loi ARN ARN thng tin (iARN; mARN) c cu to t mt mch poliribonucleotit ph bin c t khong 600-1500 ribonucleotit. Phn t mARN c mt b ba m u (AUG), sau n b ba quy nh axit amin th nht, th hai... cui cng l b ba kt thc (UAA, UGA, UAG). Phn t mARN c chc nng truyn t thng tin di truyn t ADN trong nhn t bo n t bo cht v trc tip tham gia tng hp protein. Hm lng mARN rt t, ch chim vi %

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ARN tng s trong t bo. ARN vn chuyn - ARN ho tan (tARN; sARN): Cu to t mt mch poliribonucleotit, c khong 75-100 ribonucleotit, khi lng phn t 26000 dalton. Hm lng tARN chim khong 10-20% ARN tng s. Phn t tARN c cu trc xon to ra cc thy trn, mt thy mang b ba i m, mt thy nhn bit enzyme gn vi axit amin mt u tARN. Mt u mt mang axit amin kt thc l ACC, cn u mt kia l GGG. Chc nng ca tARN l kt hp vi axit amin tng hp protein. Phn t tARN c 2 chc nng trong s tham gia tng hp protein l tip nhn v lin kt.

Hnh 1.12. M hnh cu trc khng gian v cu trc phn t ca tARN

ARN ribosom chim phn ln trong t bo, khong 80% hm lng ARN tng s. Cc phn t rARN cng vi protein cu to nn cc ribosome. Bn cht ho hc ca ribosome l nucleoprotein, gn 36% protein v 64% rARN. 4.1.2. Cu trc ca ARN Cc loi ARN u ch c mt chui poliribonucleotit, mi ribonucleotit gm 3 thnh phn (H3PO4; C5H10O5; baz nit: A, U, G, C). Cc ribonucleotit lin kt vi nhau bng mi lin kt phosphodieste, to thnh chui poliribonucleotit, c chiu 5' 3'. Tt c cc loi ARN u c tng hp t khun mu ca ADN v ch c cu to si n. Phn t ARN ca virut l genome ca chng, c chc nng duy tr v truyn t thng tin di truyn. Ring cc retrovirut mang ARN si kp. 4.2. C ch phin m 4.2.1. Phin m sinh vt nhn s 4.2.1.1. ARN-polimerase ca sinh vt nhn s E. coli, ARN-polimerase c h s lng 11S - 13S v khi lng phn t 500.000 dalton; t enzyme ARN polimerase nhn t sigma ( ) c th kt hp

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vi enzyme li khc.

Hnh 1.13. Enzyme ARN -polimerase

Nhn t ng vai tr quan trng trong vic tng hp ARN, n gip cho enzyme li c th khi u tng hp ARN ti nhng im c th - gi l im khi u (promotor), nu thiu nhn t th qu trnh tng hp ARN s xy ra im ngu nhin trn phn t ADN. on nhn bit (recognition sequence) gm 35 cp baz nm trc im khi u promotor. on ARN-polimerase nhn bit l on gm 7 nucleotit c gi prinow, nm cch im phin m 5 - 6 baz. Hp prinow trn si i khun (antitemplate) c cng thc chung l: 5' TAT phun AT purin 3'. 4.2.1.2. Cc giai on ca qu trnh phin m Qu trnh phin m ca Prokaryot bao gm 3 bc: khi u, ko di v kt thc.

1) ARN-polimerase li cng nhn t nhn bit v bm vo trnh t khi ng (promotor). ADN polimerase trt dc gene (3' - 5') xc tc bin tnh cc b 2 si ADN l ra si khun tng hp ARN.

2) Qu trnh tng hp ARN bt u v si ARN c sinh ra theo chiu 5' - 3'.

3) on kt thc ra tn hiu cho ARN-polimerase dng phin m. on kt thc gm 2 phn: phn c t l GC cao v mt loi baz T trn si antitemplate.

4.2.1.3. c im Sn phm ca qu trnh phin m l si n ARN. ADN dn xon cc b, ch c 1 si lm khun tng hp ARN v c gi l si c ngha (strand sense). Nguyn liu l cc ribonucleotit triphotphat : ATP, GTP, CTP, UTP gi chung l NTP, enzyme xc tc l ARN-polimerase. ARN c tng hp theo chiu 5' - 3', enzyme ARN-polimerase di chuyn theo chiu 3' - 5' trn si strand sense. 4.2.2. Phin m sinh vt Eukaryot 21

4.2.2.1. ARN polimerase ARN-poIimerase I: enzyme khng b c ch bi a-amanitin (cht c ch

tng hp ARN), chuyn trch vic phin m cc gene tng hp rARN (28S, 58S v 18S).

ARN-polimerase II: enzyme b c ch bi - amanitin, n cn cho tng hp mARN.

ARN-polimerase III: enzyme b c ch bi - amanitin nng cao, phin m tng hp cc tARN 4S, rARN 5S.

4.2.2.2. Cc on kim sot phin m Hp TATA hoc hp Goldberg - Hogness v tr cch cp baz u tin c phin m khong 30 cp baz v pha trc c coi l on khi u. on kt thc cn nghin cu t, on kt thc khng di hn 21 baz, nm men c trnh t l: TTTTATA. 4.2.2.3. Cc giai on ca qu trnh phin m Eukaryot

Giai on khi ng: chu s kim tra ca mt trnh t c bit l "hp TATA" (TATA box) nm trc v tr bt u phin m khong 25 - 35 nucleotit. ARN-polimerase II hot ng phin m nh nhiu nhn t TF c bn cht l protein (trascription factor) cho php khi ng s phin m.

Giai on ko di: phn t ARN c tng hp t mch khun ADN. Qu trnh ny nh nhn t TF IIS.

Giai on kt thc: phin m kt thc trc im gn ui poli A rt xa. Kt thc phin m c lin quan n cu trc kp tc tip ngay sau trnh t giu GC.

4.2.2.4. Din bin qu trnh phin m 1) Gn chp: khi pre mARN ang to ra di 20 - 30 baz th u 5'P ni

thm 7-methylguanosine lin kt 5'P - 5'P. Bn phin m u tin l tin mARN gm c exon v intron.

2) Thm ui poli A: mt on ngn ca mARN b ct ra v cc baz adenine ni vo thnh ui poli A.

3) Splicing: ct ri cc intron v ni cc exon nh phc hp ribonucleoprotein (SnRNP) ca nhn, to cu trc khng gian thun tin cho cc u exon gn nhau v xc tc phn ng ct ni hnh thnh ARN trng thnh.

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Hnh 1.14. S cc giai on ca qu trnh phin m Eukayot 4.2.2.5. c im ca s phin m Eukaryot

ARN-polimerase II chu trch nhim tng hp mARN, cn 2 loi enzyme khc tng hp rARN v tARN.

mARN, rARN v tARN cha thng tin dng bn sao ca mt gene. Qu trnh phin m phc tp, u 5' ca mARN c gn thm 1 "chp"

(cap: chp, chp n) l 7-methylguanosine, cn cui mARN c "ui" poliadenine di 100 - 200 A. Chp - Methylguanosine v ui poliA c tc dng bo v ARN, ngoi ra ui poliA cn l tn hiu nhn bit m kt thc trong qu trnh dch m.

Bn phin m u tin l tin mARN (premessager ARN-Pre-ARN) bao gm c cc on intron v exon, sau sau cc intron b ct b to thnh mARN trng thnh.

THO LUN 1. Phn bit cc khi nim: genome, genome nhn, genome ti th, genome

lp th. 2. c im v vt cht di truyn ca Virut, Prokaryot, Eukaryot. 3. Vn cu trc ca h gene ti th. Nhng ng dng nghin cu h gene ti

th cy trng v ngi. 4. c im cu trc v chc nng ca ADN v ARN. 5. C ch ti bn ca ADN. Ti bn ADN Virut, Prokaryot, Eukaryot. Ti

bn ADN v ti sao trong ti bn ADN li cn phi c primer? Ti sao primer l mt trnh t ARN?

6. c im ca c ch phin m Prokaryot, Eukaryot. Vn phin m 23

ngc v ng dng trong thc tin.

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Chng 2: C IM CU TRC GENE CA SINH VT PROKARYOT V EUKARYOT

Tm tt: Nhng thao tc trn gene c da trn c s nhng hiu bit v c im cu trc ca gene. Gene nhm sinh vt Prokaryot v nhm Eukaryot c nhng c im cu trc c trng. Gene ca a s sinh vt Prokaryot c cu trc lin tc, bao gm cc trnh t m ho axit amin; gene ca sinh vt Eukaryot c cu trc gin on (gene phn mnh) gm cc intron v exon xen k nhau. c im cu trc ny quyt nh s khc nhau gia qu trnh phin m Prokaryot v Eukaryot. Cc trnh t nh promotor, operator, enhancer, yu t di truyn vn ng (TGE), on xen (IS), trnh t lp li CEN, TEL c cu trc v chc nng xc nh trong h thng di truyn ca t bo. Hin tng bin tnh, hi tnh ca ADN, nhit chy Tm cng l c s ca mt s k thut di truyn nh lai phn t, PCR, v mt s k thut khc. Ni dung ca chng gm 3 vn c bn : (1). c im cu trc gene ca sinh vt Prokaryot; (2). Cu trc phn on gene ca sinh vt Eukaryot; (3). Mt s trnh t ADN.

1. C IM CU TRC GENE PROKARYOT Gene ca a s sinh vt Prokaryol gm nhiu cistron (a cistron), trnh t promotor, operator. Gene cu trc ca Prokaryot c cu trc lin tc, bao gm nhng on m ho axit amin. Promotor l trnh t trn phn t ADN c i lc vi enzyme ARN-polimerase, c chc nng khi ng qu trnh phin m. Operator l trnh t c i lc vi protein c ch, nu operator gn vi protein c ch s ngn cn hot ng ca ARN-polimerase v c ch qu trnh phin m. Mi cistron cha thng tin ca mt loi chui polipeptit v cng l nhn t iu khin qu trnh tng hp chui polipeptit thng qua qu trnh phin m v dch m.

2. CU TRC PHN ON GENE EUKARYOT 2.1. Cu trc gene phn on (gene phn mnh) Mt gene Eukaryot c cu trc bao gm on tng cng (enhancer) on khi ng (promotor), intron v k nhau, cui cng l on kt thc. ho (Noncoding Sequences) c gi l intron hay gi l on xen (Intervening 25

Sequences) (IS) xen vo v lm gin on cc on m ho (Coding Sequences) c gi l exon.

Hnh 2.1. Cu trc gene phn on Eukaryot Nhng gene c cu trc gm exo c gi l gene khm (mosaic gene). Hin nay, cha r chc nng ca intron, tuy nhin c gi thuyt rng, intron c vai tr nh l cc khong trng xen gia cc exon to thun li cho s ti t hp gia cc exon. Th nghim ca Pierre Chambon (Php) pht hin cu trc ca exon - intron ca gene ovalbumine g. Ovalbumine l protein lng trng trng. gm mt chui polipeptit c 386 axit amin (tp hp trong ng dn trng thi k g mi trng). Ngoi vic tch mARN ca ovalbumine, s dng enzyme sao m ngc to ra cc bn sao ca mARN si n c gi l cADN v to thnh cADN si kp. So snh ADN h gene nhn vi cADN khi gii trnh t cho php pht hin ra intron v exon. Kt qu ovalbumine trng g c 7 intron, 8 exon. 2.2. Phng php pht hin cc on intron v exon 2.2.1. Phng php so snh trnh t gene v trnh t cADN

Bc 1: 1. Tch chit ADN h gene. 2. Phn lp gene bng k thut PCR hay enzyme ct hn ch. 3. To dng ADN ti t hp, nhn dng v tch dng gene. 4. Gii trnh t gene. Bc 2: 1. Tch mARN trong t bo cht (hm lng mARN rt ln, mARN ca ovalbumine chim ti 50% ARN trong cc t bo ny). 2. S dng enzyme sao m ngc tng hp cARN. 3. Gii trnh t cARN. Bc 3: So snh trnh t ADN h gene vi trnh t cADN xc nh c cc on intron v exon.

2.2.2. Phng php lai phn t Pht hin intron v exon cn c tin hnh theo phng php lai phn t

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v kim tra bng knh hin vi in t. 1. Tch ADN h gene v mARN t bo cht. 2. Phn lp gene. 3. Lai phn t gia ADN v mARN s xc nh c intron v exon.

Vn t ra l khi phin m tng hp mARN th cc on khng m ho intron c c sao khng ? Ngi ta thy bn sao ca ADN c c exon v intron chnh l tin mARN (Pre-mARN), cn gi l ARN khng ng nht trong nhn (hn-ARN: heteroge nevusnucliar ARN). Loi hn-ARN ch c pht hin sinh vt nhn chun.

3. MT S TRNH T ADN 3.1. Promotor Thc cht ca khi u phin m l quan h tng tc gia ARN-polimerase vi trnh t promotor. Khi ARN-polimease gn vo promotor th qu trnh phin m c thc hin v kt qu s to phn t mARN.

Phn ln cc promotor E. coli v cn bn c cng mt cu trc theo s sau:

ADN

sao m hn-ARN tin mARN

processing

ct ni mARN

-35 bp -10 bp +1

TTGACA mARN TATAAT

Nu baz u tin c phin m ra mARN (thng l A) c nh s +1 v tt c cc baz pha 5' hay pha trc n khng c phin m c ghi l (-). Ngay trc +l c 6 baz thng l trnh t TATAAT xung quanh -10, gi l trnh t nht tr (consensus sequense), c th gi pribnow box. Xung quanh trnh t -35 c trnh t nht tr TTGACA. C hai trnh t phi hp vi nhau cho php ARN-polimerase gn vo promotor v qu trnh phin m v dch m bt u. Promotor Eukaryot nm pha trc im xut pht ca mARN. Hp TATA, nh hng cho ARN-polimerase bt u phin m, nm khong di

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30 bp (ng vt c v) v 6-120 bp (nm men). Trc hp TATA c hai trnh t tng ng pha trc khong 40 bp l CCAAT v 110 bp l trnh t giu GC.

-110 bp -40 bp -30 bp +1

GGGCGGG CCAAT TATA mARN

S thay i hp TATA lm gim tc phin m. Hiu qu ca tt phin m o bng s thay i tng baz trong promotor. Hp TATA v cc trnh t pha trc phi c nhn bit bi cc protein iu ho, chnh cc protein ny gn vi cc im nht nh trn chng v hot ho s phin m. 3.2. Enhancer (trnh t tng cng) Enhancer l trnh t c tc ng cis (u pha) c chc nng lm tng tc phin m, enhancer c hiu qu ngay c khi cch xa vi nghn cp baz v chng hot ng bt k hng no, d l trc hay sau promotor. Trnh t tham gia iu ho - cis c cu trc gm 2 phn i xng nhau:

AGGTCA TGACCT TCCAGT ACTGGA

Enhancer c t chc gm mt dy cc trnh t c tc ng cis nhn bin cc nhn t trans - nhn tprotein tham gia iu ho biu hin gene. Nhn t trans gm 2 vng cu trc chc nng: vng gn trans vi ADN v vng tc ng ln phin m. Cc nhn t trans c 4 kiu tc ng nh:

"Ngn tay km" (zine-finger) "Xon-vng-xon" (hehx-turn-hehx) "Xon-nt-xon" (hehx-loop-helix) "Dy ko leucine" (leucine-zipper)

Gia trnh t cis v nhn t trans thng c cc tng tc c hiu do nhng lin kt yu hnh thnh gia cc phn t ca nhn t trans vi cc baz ca trnh t cis. Cc trnh t cis thng tip nhn protein iu ho (nhn t trans) gm 2 tiu n v (dimer). Nhn t trans c th c 4 nhm cu trc, chng lun gn ln cis. iu ho bng cch bin i nhim sc cht hay ADN. Nhim sc th tn ti nhng vng "nhy cm" tng ng vi cc gene hot ng. Cc vng nhy cm ny d tip xc vi enzyme sao m. Tm li, cc gene ca Eukaryot c hot ho bi hai trnh t ADN c tc ng cis l promotor v enhancer. Chng c nhn bit cc nhn t protein 28

c Lc ng trans. Cc nhn t trans ny cho php ARN-polimerase khi ng phin m v t tc phin m ti a. 3.3. Yu t di truyn vn ng (Transposable Genetic Element-TGE) Gene nhy (Jumping genes -JG) Gene nhy (Jumping genes) cn dc gi l yu t di truyn vn ng (TGE) ln u tin c pht hin bi Barbara Mc Clintock khi nghin cu ng vo nhng nm 40 ca th k XX v b c nhn gii thng Nobel 1983. TGE l nhng on ADN c bit xen vo mt hoc mt s v tr trong h gene, to nn nhng bin i di truyn. Loi TGE n gin nht l cc on xen (Insertion Sequence - IS ). IS cn thit cho qu trnh xen ADN vo NST, cho qu trnh chuyn TGE t v tr ny sang v tr khc trong h gene. Cu trc ca IS ging nhau nhng sinh vt khc nhau. Ngi ta pht hin c E. coli mt on xen IS1 cha 720 bp (base pair), nm gia on o ngc IR (24 bp)

Hnh 2.2. on xen IS1 E. coli

E. coli cn pht hin ra gene nhy mang tn Tn 1681 (Transposase) gm 2 on xen IS1 hai u v 1 gene khc di 552 bp quy nh c t chu nhit gy a chy.

Hnh 2.3. Gene nhy Tn 1681 E. coli

Gene nhy Tn E. coli c th chuyn v tr v xen vo mt nhim sc th khc theo c ch c biu din bng m hnh hnh 2.4. 3.4. Bin tnh v hi tnh Hai mch ADN lin kt vi nhau bng nhng lin kt hyro. Khi nhit ln ti 80-950C th hai mch ADN tch nhau, c gi l hin tng bin tnh ca ADN.

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Nhit lm 2 mch ADN tch ri nhau c gi l im chy (melting point). im chy c tnh gia ng cong OD hnh Sigma. im chy k hiu Tm. ADN c hm lng G-C cao th im chy cao hn. ADN c 60% l G- C th Tm = 950C.

Hnh 2.4. C ch xen ca gene nhy Tn 1681 E. coli

Khi nhit h xung v tr li bnh thng, th hai mch n li lin kt vi nhau to thnh ADN mch kp. Hin tng ny c gi l hi tnh (Renaturation). 3.5. Trnh t lp li 3.5.1. Khi nim Hin tng trong cc phn t ADN c cc trnh t baz ngn c lp li nhiu ln gi l trnh t lp li. V d: ATAT GCGTCCATATCGCGCTATATGCGTAGC S khng ng nht ca ADN Eukaryot th hin r khi thc hin phn ng ti hp ADN (Reassociation).

ADN b ct nh bi enzyme gii hn. ADN c bin tnh. Hi tnh: Cc trnh t b sung d ti t hp vi nhau, t nhn bit c

cc trnh t lp li.

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Hnh 2.5. ng cong Tm

Trnh t CEN Cc trnh t lp li cao CEN l nm cc tm ng (Centromere) ca nhim sc th. nm men Saccharomyces gm cc trnh t CEN. - Trnh t u c 9 cp baz TCACATGAT ( u 5') - Trnh t gia 80 - 90 bp, rt giu A - T (>90%) - Trnh t 11 cp baz u 3': TGATTTCCGAA

Trnh t TEL (Telomere - u mt NST) Trnh t TEL c tnh lp li cao, giu A v C: CCCCACACACCA (nm men) v CCCTAA (ngi). u mt NST giu G gp li thnh nhng hnh kp tc c cu trc bc IV. Cu trc ny bo v u mt NST c th b ct bi nuclease, gi cho u mt khi mt cc trnh t m ho. 3.5.2.ng dng nghin cu trnh t nucleotit lp li Gn y mt loi ch th phn t mi c gi l vi v tinh - microsatehtte (Litt and Luti, 1989) hoc trnh t nucleotit lp li n gin - Simple sequence repeat (Tautz 1989, Weber and May, 1989) c m t sau khi tin hnh phn ng tng hp ADN nh polimeraza v in li trn gel poliacrylamit ngi ta c th pht hin loi a hnh ny. Trnh t hai nucleotit lp li rt phong ph trong b gene ca ngi cng nh ng thc vt. Tanksley v cng s, 1993 khi nghin cu hin tng a hnh ca c( vi v tinh la cho thy 2 nucleotit lp li (GA)n hoc (GT)n xut hin sau mi mt on ADN di 150 Kb, tng s 2 nucleotit ny chng 3000. y l ngun phn t ADN a hnh phong ph i vi vic lp bn gene loi cy trng ny. Yanagisawa v cng s (1994) nghin cu du vn ADN ca 47 ging

31

u tng thng qua cht d Oligonucleotit nhm pht hin trnh t ADN lp li n gin v cho bit hin trng a hnh th hin r nht l khi dng Oligolucleotit (AAT)6 lm on d ADN tt c ging u tng th nghim u phn bit vi nhau bi on d . Khi s dng ba on d ADN khc nhau nh (CT)8, (GAA)5 v (AAGG)4 th khng pht hin c cc bng ADN a hnh t cc ging u tng thuc Subgeneus Soja (Glicine max v Glicine Soja), tri li cc bng ADN a hnh xut hin trong s cc ging thuc Subgeneus, Glicine. Kt qu nghin cu chng t G. max v G. Soja gn gi nhau v cu trc b gene.

THO LUN 1. Phn bit cu trc ca gene sinh vt Prokaryot v Eukaryot. Vn v

c im ca gene sinh vt Eukaryot. 2. Phng php xc nh cc on intron v exon trong gene ca sinh vt

Eukaryot. Cho v d. 3. Phn bit khi nim v gene nhy v on xen. C ch xen ca gene nhy. 4. Trnh by cc khi nim: trnh t lp li, bin tnh v hi tnh.

ng dng ca cc k thut ny trong phn tch sinh hc phn t.

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Chng 3: MI LIN H GIA ADN, ARN, PROTEIN Tm tt: Thng tin cu trc ca phn t protein, tARN, rARN c m ho trong gene bng trnh t cc nucleotit. Trnh t nucleotit trong gene m ho cho chui polipeptit c xc lp bng thc nghim v th hin trong bng m di truyn. M di truyn l m b ba nucleotit (codon), s thay i trong codon c th dn n s thay i trong chui polipeptit v t bin tnh trng. Nhng c im v cu trc v chc nng ca protein v mi lin h gia ADN, ARN, protein v s iu ho biu hin gene cng l c s quan trng xy dng k thut biu hin gene. Ni dung ca chng gm 4 vn c bn: (1). Thng tin sinh hc v mt m di truyn; (2). Protein; (3). Tng hp protein; (4). iu ho biu hin gene.

1. THNG TIN DI TRUYN V MT M DI TRUYN Thng tin quy nh cu trc mt loi protein, mt loi tARN, rARN c gi thng tin di truyn. Thng tin di truyn c cha ng v c m ho trong phn t ADN bi trnh t cc nucleotit. Hin tng 3 nucleotit k tip nhau trong gene quy nh mt axit amin trong chui polipeptit c gi l s m ho b ba. Cn mt b ba c th xc nh cho mt axit amin c th ( v d: UGU: serine) c gi l b ba m ho (codon - code). Phn t tARN c mt b ba b sung vi b ba m sao ca mARN c gi l b ba i m (anticodon) ADN cha thng tin di truyn, l thng tin quy nh cu trc mt loi protein, rARN v tARN. Trnh t ca cc baz trong ADN quyt nh trnh t ca axit amin trn protein tng ng. Phn t ADN c cu to t 4 loi nucleotit, v protein li c cu to t 20 loi axit amin, nu, mt nucleotit quy nh 1 loi axit amin s to ra 4 t hp m; cn hai nucleotit quy nh 1 loi axit amin s to ra 42 = 16 t hp m b 2, nh vy vn cn thiu 4 loi axit amin cha c xc nh bi cc m b hai. V ba nucleotit quy nh 1 loi axit amin s to ra 43 = 64 t hp m b 3. Ngi ta chng minh bng thc nghim: m di truyn l m b ba. i tng l gene rII phage T4. Trong th nghim ngi ta to t bin mt (-) v thm (+) mt cp baz trn ADN bin i ADN thnh phn axit amin ca protein. Gi s phn t mARN ch c thnh phn CAG v trong phn t protein ch c mt loi axit amin:

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mARN CAG CAG CAG CAG CAG Protein a1 a1 a1 a1 a1 - Nu t bin lm mt C (-) v tr m s 2 trn mARN: mARN CAG AGC AGC AGC AGC protein a1 a2 a2 a2 a2 - Nu t bin (+) thm G vo gia b ba th 2: mARN CAG AGC GAG CAG CAG CAG protein a1 a2 a3 a1 a1 a1 Cc s liu thc nghim thu c trn th nghim rII thc khun T4 chng t lp lun trn l ng. Vy m di truyn l m b ba nucleotit. 1.1. Gii m di truyn Nm 1961 M. Nirenberg s dng h thng v bo gii m di truyn. H thng v bo c chit t E. coli c cha ribosome, ARN, enzyme aminoacylsynthetase, mARN, cc axit amin v mt s ph gia khc. Phn ng din ra trong vi pht ri dng li, nu b sung thm mARN th vic tng hp li tip din. Nu mARN nhn to ch ton uraxin th chui polipeptit c tng hp ch c loi axit phenylalanine, nu mARN ch ton adenine th chui polipeptit c tng hp ch c loi axit lisine; nu mARN ton cytosine th chui polipeptit c tng hp ch c loi axit proline. T c th suy ra b ba UUU quy nh axit amin phenylalanine; AAA : lisine; CCC : proline. Bng phng php ngi ta tm ra 61 b ba m ho. Ba b ba cn li l UAA, UAG, UGA lm nhim v nhn bit tn hiu kt thc qu trnh tng hp chui polipeptit, cn gi l b ba v ngha (non sense) v chng khng quy nh axit amin. 1.2. Cc c tnh ca m Thng tin c c theo tng cm ba nucleotit mt cch lin tc khng ngt qung.

- Thng tin c c theo mt chiu, bt u t mt im xc nh. - M di truyn mang tnh ph bin (umversal). - M di truyn mang tnh thoi ho (degenerate), tr 2 ngoi l AUG v

UGG. - M di truyn mang nhng b ba khi u (AUG) u 5'. Cc b ba kt

thc UAG, UAA, UGA.

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Bng mt m di truyn (m sao mARN)

U C A G UUU. Phe UCU Ser UUC Phe. UCC Ser UUA Leu UCA Ser

U

UUG Leu UCG Ser

UAU Tir UAC Tir UAA KT UAG KT

UGU Cys UGC Cys UGA KT UGG Trp

U C A G

CUU Leu CCU Pro CUC Leu CCC Pro CUA Leu CCA Pro

C

CUG Leu CCG Pro

CAU His CAC His CAA Gin CAG Gin

CGU Arg CGC Arg CGA Arg CGG Arg

U C A G

AUU Isoleu ACU Thr AUC Isoleu ACC Thr AUA Isoleu ACA Thr

A

AUG Met ACG Thr

AAU Asn AAC Asn AAA Lis AAG Lis

AGU Ser AGC Ser AGA Arg AGG Arg

U C A G

GUU Val GCU Ala GUC Val GCC Ala GUA Val GCA Ala

G

GUG Val GCG Ala

GAU Asp GAC Asp GAA Glu GAG Glu

GGU Gli GGC Gli GGA Gli GGG Gli

U C A G

M khi u AUG c 2 chc nng: va to ra s bt u dch m (m m u) va m ho axit amin. (Mt vi trng hp m m u khng phi l AUG m l UGU iu khin axit amin formyl methionine i vo u chui polipeptit). M kt thc (codon v ngha - nonsense):

UAA (ochae) Tn hiu kt thc UGA (opal) qu trnh tng hp protein UAG (amber)

- Tnh linh hot Trong t bo c nhng loi tARN c th ng thi c mt s b ba khc nhau. Phn tch trnh t tARN cho thy rng mt s tARN c cu trc inosine l mt trong cc baz ca cm m i (anticodon). c im ca inosine c th hin hnh 3.1. Phn tch trnh t cho thy u 5' ca codon i m (b tr vi baz th ba ca cm m) khc vi 2 baz kia, c kh nng kt hp vi mt s baz trn u 3' ca cm m. Hin tng ny cn c gi l tnh "linh hot" trong kt cp baz v tnh linh hot l c gii hn:

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Hnh 3.1. Cu trc ca inosine v guanine

Baz trn u 5' ca anticodon Baz trn u 3' ca cm m G kt cp vi U hoc C C kt cp vi G A kt cp vi U U kt cp vi A hoc G I (Inosin) kt cp vi A, U hoc G

Hnh 3.2. Tnh linh hot ca m di truyn

1.3. t bin v m di truyn Mi lin quan gia t bin v m di truyn c th hin 4 kiu t bin c bn. 1.3.1. t bin nhm ngha Mt cp baz b bin i dn n thay i cm m tng ng trn mARN v lm thay i 1 axit amin trn polipeptit.

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1.3.2. t bin v ngha

3' AGT CAA GGT TGC CAT 5' 1 2 3 4 5 t bin thay th b ba th 4 (G-C b thay th bi A-T) 3' AGT CAA GGT TAC CAT 5' Thay i mARN 5' UCA GUU CCA AUG GUA 3' mARN

Bin i mt cp baz lm xut hin mt codon v ngha dn n xut hin tn hiu kt thc chui polipeptit lm cho chui polipeptit c tng hp ngn (t axit amin) hn bnh thng.

3' AGT CAA GAT TGC CAT 5' 1 2 3 4 5 t bin mt cp baz G-X b ba th 3 3' AGT CAA ATT GCC AT 5' Xut hin m kt thc UAA mARN 5' UCA GUU UAA CGG UA 3' mARN

1.3.3. t bin cp baz b ba quy nh axit amin 1m xut hin codon mi (codon thoi ho) khng nh hng n chui polipeptit Mt loi axit amin c th c quy nh bi mt vi b ba: v d GUU, GUC, GUA, GUG cng quy nh axit amin valine. Trng hp bnh thng:

3' AGT CAA GGT TGC CAT 5' 1 2 3 4 5 5' UCA GUU CCA ACG GUA 3' mARN Scrine - Valine - Proline - Threonie - Valine.. Protein

Trng hp t bin.

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1.3.4. t bin dch khung

3' AGT CAA GGT TGC CAT 5' 1 2 3 4 5 t bin thay th b ba th 2 (A-T b thay th bi G-X) 3' AGT CAG GOT TAC CAT 5' Thay i mARN 1m BD i GUU thnh GUC 5' UCA GUC CCA AUC GUA 3' mARN ... Serine - Valine - proline - Threonie - Valine..... Protein (GUU v GUC cng quy nh valine)

Hin tng mt hoc thm mt cp baz dn n t hp li cc codon v lm thay i thnh phn axit amin ca chui polipeptit.

Trng hp bnh thng:

3' AAC ACA CAG GTT CAA AGG AGC 5' Gene Phin m 5' UUG UGU GUC CAA GUU UCC UCG 3' mARN Dch m - Leucinne - Cysteine-Valine - Glicine - Valine - Tryptophane - Serine Protein

Trng hp t bin dch khung:

3' AAC ACA CAG GTT CAA AGG AGC 5' Gene t bin mt cp C-G b ba th 3 3' aAC acAn agg ttc aaa Gga gc 5' Gene t bin Phin m 5' UUG UGU UCC AAG UUU CCU CG 3' mARN Dch m - Leucinne - Cysteine - Serine - Lisine - Tryptophane - Proline Protein

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Nh vy chui polipeptide c thnh phn axit amin: - Leucinne - Cysteine-Valine - Glicine - Valine - Tryptophane - Serine Bin i thnh: - Leucinne - Cysteine - Serine - Lisine - Tryptophane - Proline.

2. PROTEIN 2.1. Chc nng ca protein trong t bo Protein chim khi lng ln trong t bo v tham gia vo hu ht cc qu trnh sinh hc. Thnh phn cu to nn cc b phn ca t bo, quy nh nhng c im v hnh thi v cu to ca t bo v c th. Protein enzyme c kh nng xc tc ln v tnh c hiu cao. Khng th: chng li cc tc nhn xm nhp vo t bo v c th. Tc nhn gy min dch (khng nguyn) xm nhp vo c th ng vt c xng sng s kch thch s sn sinh ra khng th. Protein vn ng (myosin), cm ng. 2.2. Cu trc ca protein Axit amin l n v cu to nn protein, c khong 20 loi axit amin cu to nn cc phn t protein khc nhau. Axit amin c cu to chung nh sau:

Da vo din tch ca chng cc aminoacit c th chia lm 4 nhm:

Nhm I. Cc axit amin c tnh kim: lisine, argimne, histidinc. pH ca t bo nhm amine (NH)2 nhnh bn b ion ho thnh NH3, nn chng mang in tch dng.

Nhm II. Cc axit amin mang tnh axit, v nhnh bn b oxy ho thnh COO-, bao gm: aspartic v glutamic.

Nhm III. Cc axit amin trung tnh k nc (khng mang in tch) nh: alanine, valine, leucine... c nhnh bn mang cc nhm k nc.

Nhm IV. Cc axit amin trung tnh phn cc c nhnh bn nhm OH to cc lin kt hyr a nc.

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Cc axit amin lin kt vi nhau bng mi lin kt peptit. Protein c cu trc theo nhiu bc:

Bc I: chui polipeptit c dng mch thng. Bc II: chui polipeptit c dng mch xon . Bc III: chui polipeptit c dng mch hnh cu. Bc IV: t chc protein c nhiu chui polipeptit :

Hnh 3.4. Lin kt to chui polipeptit

Hnh 3.5. S cc bc cu trc ca protein Mt phn t protein ch c mt chui polipeptit (cu trc bc I, II, III) hoc gm nhiu chui polipeptit (cu trc bc IV).

40

41

Hnh 3.7. a, b: Cu trc bc II; c, d, e, g: Cu trc bc III; m. n, p, q: Cu trc bc IV ca protein.(theo Phan Vn Chi, 2002)

3. QU TRNH SINH TNG HP PROTEIN 3.1. Cc yu t tham gia tng hp protein 3.1.1. Cc phn t ARN Mi phn t mARN mang thng tin xc nh trnh t ca mt polipepetit, thng tin ny c phin m t ADN. S lng loi tARN bin ng theo loi t 30 - 40 loi (Prokaryot), 50 loi (Eukaryot), nhng cu trc ca cc loi tARN rt ging nhau. Chc nng ca tARN c thc hin nh cc enzyme c bit l cc aminoacyl-tARN-synthetase. C 20 loi aminoacyl-tARN-synthetase tng ng vi 20 loi axit amin. Qu trnh gn axit amn vi tARN vi s tham gia ca enzyme ny l qu trnh tiu tn nng lng v tri qua 2 bc:

Enzyme nhn bit v gn vi 1 axit amin c hiu Enzyme + a.a + ATP enzyme - aminoacyl - AMP + P.P

Axit amin c chuyn t phc hp enzyme aminoacyl sang tARN tng ng. Enzyme - aminoacyl - AMP + tARN tARN- aminoacyl + AMP + E 3.1.2. Ribosome (RBX)

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Ribosome (RBX) c cu to t rARN v protein to thnh ribonucleoprotein. RBX c 2 tiu phn (tiu n v); tiu n v ln cha 1 phn t rARN ln v tiu n v nh cha 1 rARN nh. RBX Prokaryot v Eukaryot c bn ging nhau.

Hnh 3.8. Ribosome

3.2. Cc giai on ca qu trnh tng hp protein Qu trnh sinh tng hp protein trong t bo tri qua hai khu: phin m v dch m (hnh 3.9). y ch trnh by khi qut cc giai on ca qu trnh dch m. 3.2.1. Khi ng Giai on c s tham gia ca nhiu nhn t protein - nhn t khi ng (IF - Initiation Factor); Prokaryot c 3 nhn t IF, cn Eukaryot c 6 nhn t IF. Du hiu khi ng l codon AUG (m ho methyonine). C 2 loi tARN mang a.a Met c kh nng kt hp vi codon AUG:

(1) tARNmet : kt hp vi Codon AUG gia mARN (2) tARimet : kt hp vi AUG khi ng, gn a.a methyonine u tin ca

chui polipeptit. Cc bc ca giai on khi ng: Aminoacyl - tARN - synthetase chuyn bit (methionyl - tARN - synthetase) gn 1 phn t met vo mt u ca tARNmet to thnh Met - tARNimet. Hnh thnh tiu n v nh ribosome - met - tARNimet - mARN vi s tham gia ca nhn t khi ng (IF2 - Prokaryot ; IF4 - Eukaryot). 3.2.2. Ko di Khi a.a met t vo v tr chui polipeptit bt du c tng hp. 43

Aminoacyl - tARN k tip n n xp vo ng v tr trn ribosome nh mt nhn t ko di (EF). RBX c 2 v tr chuyn bit (hnh 3.6).

Hnh 3.9. S gn ca mARN vi cc tiu phn ribosom v s dch m ca

chng A: tip nhn aminoacyl - tARN. P: gi phc hp peptidyl - tARN hnh thnh chui polipeptit. Qu trnh c lp li cho n khi xut hin du hiu kt thc dch m. 3.2.3. Kt thc Khi nhn t (TF) kt thc nhn bit c du hiu kt thc (cc codon kt

44

thc: UAG, UAA, UGA), phc hp peptidyl - tARN lp tc tch thnh tARN t do v chui polipepetit. RBX tch ra thnh 2 tiu n v v mARN c gii phng.

Hnh 3.10. Dch m tng hp protem cc ribosom trn li ni cht

4. IU HO BIU HIN GENE Ba thnh phn c bn ca s iu ho hot ng gene.

(1). Nhng tn hiu lm thay i biu hin gene. (2). iu ho biu hin gene thc hin giai on no t sao chp n dch m. (3) C ch phn t ca s iu ho biu hin gene.

4.1. iu ho biu hin gene Prokaryot Cc gene c phin m to ARN, c gi l gene cu trc, protein c dch m t mARN c th enzyme hoc khng phi l enzyme. Trong s cc protein khng phi enzyme c protein iu ho (regulatory prtotenin) chng tng tc vi trnh t ADN c hiu kim sot phin m ca gene cu trc. Gene iu khin tng hp protein iu ho gi l gene iu ho (regulatory gene), pha trc nhm gene cu trc c trnh t protnotor, ni ARN-polimerase nhn bit.

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Hnhh 3.11. Mi quan h gia AND, ARN v protein

Thc cht ca khi u phin m l quan h tng tc gia ARN - polimerase vi promotor. Khi ARN-polilmese gn vo promotor gene s to phn t mARN. 4.1.1. Cu trc ca operon S iu ha biu hin gene Prokaryot phn ln xy ra giai on phin m. C ch iu ha thng qua operon. Mi operon bao gm: nhm gene cu trc (cistron) cnh nhau v cc trnh t AND khc nhau tham gia iu ha. Operon l n v phin m gm t nht 1 promotor (trnh t khi ng) v mARN k cn m ha cho cc trnh t ca mt hay nhiu mch polipeptit. Promotor c i lc vi ARN-polimerase, mt trnh t AND ni m protein c ch (repressor protein) gn vo c gi l operator (im iu hnh), k tip l cc gene cu trc (cistron). Prokaryot thng to ra mARN a gene (policistronic), nhng mARN ca Eukaryot ch c 1 gene. Kim sot m (Negative control): S gn ca repressor protein vo operator lm cn tr s phin m ca tt c cc gene trong cng mt operon. Kim sot dng (Positive control): Cc protein cn thit cho gene biu hin gi l activator (cht hot ho), activator c th gn vi cc im khi ng (initiator site) ca operon hay gn vi enhancer site (im tng cng) s kch

46

thch s phin m ca cc gene cu trc. Cht gip cho gene phin m c gi l cht cm ng (inducer), cht c tc dng ngc li gi l cht km hm. Cc gene cm ng (inducible gene) thng thng tham gia vo cc phn ng thoi dng (kim sot m, cm ng); cn cc gene c ch (repressible gene) thng tham gia vo cc phn ng bin dng. Kh nng c th xy ra 4 kiu t hp kim sot s phin m:

m, cm ng (negative, inducible control); m, c ch (negative, repressive control), Dng, cm ng (positive, inducible control); Dng, c ch (positive, repressive control).

4.1.2. iu ho thoi dng: kim sot m, cm ng (negarive, inducible control) Cc cht dinh dng c phn hu to nng lng hoc to nguyn liu cn thit cho qu trnh tng hp. C ch iu ho y l s c mt ca c cht, dn ti tng hp cc enzyme phn hu. l operon cm ng m ho cho cc enzyme ca con ng d ho. in hnh cho kiu operon cm ng l operon lactose. Tn hiu iu ho - ng lactose. Khng c lactose gene m ho cc enzyme chuyn ho lactose khng biu hin; Khi c lactose gene m ho cc enzyme chuyn ho lactose biu hin. S iu ho xy ra hon ton mc phin m. C ch ny da vo s tng tc ca protein iu ho (nhn t km hm - repressor) vi mt trnh t ADN, promotor u 5' khng m ho ca cc gene cu trc. Repressor lin kt vi operator nm gia promotor v gene cu trc. C 2 kh nng: (1) Khng c lactose: Khi mi trng khng c lactose m ch c glucose th repressor lin kt vi operator v cc cistron khng tng hp mARN - lac.

Hnh 3.12. Mi trng khng c lactose repressor lin kt vi operator v cc

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cistron khng phin m (2) C lactose:

Hnh 3.13. Mi trng c lactose inductor gn vi repressor v cc cistron phin m

Khi mi trng c lactose, gi l nhn t cm ng (inductor). Inductor gn vi repressor dn n s thay i cu trc khng gian ca repressor lm cho repressor khng cn i lc vi operator dn n qu trnh phin m ca cc cistron bt u v cc enzyme chuyn ho lactose c tng hp. 4.1.3. iu ho bin dng: m, c ch (negative, repressive control) Bin dng (anabolism) l qu trnh tng hp nn cc cht cn thit cho t bo (nh cc axit amin), l operon km hm lin quan n con ng ng ho - in hnh l operon - triptophan. Qu trnh tng hp tryptophan bt u t tin cht chorismique axit, tri qua 5 giai on k tip do 5 enzyme xc tc. H thng tng hp amino axit tryptophan E. coli do operon kim sot m, c ch. Khi mi trng khng c tryptophan t bo tng hp tryptophan nh tch cc sn xut enzyme chu trch nhim tng hp tryptophan. Mi trng c tryptophan th enzyme khng c sn xut na. Nhn t km hm repressor khng c i lc vi operator. Axit amin c trng kt hp vi repressor s lm thay i cu hnh ca repressor.

Khi mi trng c tryptophan, repressor kt hp vi tryptophan gn ln operator lm cho cc gene cu trc ca operon khng phin m.

Mi trng thiu triptophan, repressor khng gn c vo operator cc gene cu trc ca operon phin m.

4.2. iu ho biu hin gene Eukaryot B gene ca Eukaryot c kch thc ln, phn t ADN c nn trong nhn t bo. C ch iu ho biu hin gene c th xy ra 5-6 mc khc 48

nhau v s iu ho biu hin gene Eukaryot c mt s c im c bn sau: Prokaryot, cc gene iu ho v promotor nm gn nhau; cn Eukaryot cc gene iu ho t khi nm gn cc promotor do chng kim sot. Cc enhancer l nhng trnh t cng nm trn mt phn t vi cc promotor, c th c hng trm bp pha trc hoc sau m chng kch thch. Trnh t iu ho 5' pha trc promotor ca Eukaryot c th di ti hng chc kb. C nhiu kiu iu ho dng cc nhn t tc ng trans l cc protein. S iu ho hot ng gene Prokaryot phn ln p li t nhng tn hiu bn ngoi; cn Eukaryot ch yu l tn hiu bn trong. Promotor Eukaryot nm pha trc im xut pht ca mARN. Hp TATA, nh hng cho ARN-polimerase bt u phin m. S thay i hp TATA lm gim tc phin m. Hiu qu ca tc phin m o bng s thay i tng baz trong promotor. Hp TATA v cc trnh t pha trc phi c nhn bit bi cc protein iu ho, chnh cc protein ny gn vi cc im nht nh trn chng v hot ho s phin m. Enhancer l trnh t c tc ng cis (u pha) c chc nng lm tng tc phin m ng k t cc promotor, enhancer c hiu qu ngay c khi cch xa vi nghn cp baz v chng hot ng bt k hng no, d l trc hay sau promotor. Tm li, cc gene ca Eukaryot c hot ho bi hai trnh t ADN c tc ng cis l promotor v enhancer. Chng c nhn bit cc nhn t protein c tc ng trans. Cc nhn t trans ny cho php ARN-polimerase khi ng phin m v t tc phin m ti a. THO LUN

1. Trnh by v mt m di truyn. 2. t bin gene v mt m di truyn. 3. Mi lin quan gia protein v tnh chng chu. 4. c im cu trc ca protein. Qu trnh tng hp protein. 5. Phng php phn tch cu trc protein v c im cu trc ca protein. 6. iu ho biu hin gene v ng dng trong k thut di truyn.

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Chng 4: ENZYME S DNG TRONG K THUT SINH HC PHN T Tm tt: Enzyme l nhng protein hot tnh sinh hc v da vo loi phn ng xc tc m trong sinh hc phn t c th chia enzyme thnh 6 nhm khc nhau. Trong cc nhm enzyme gii hn, enzyme polimerase, enzyme ni, cc enzyme nuclease c quan tm nghin cu v ng dng trong cc lnh vc ca sinh hc phn t v k thut di truyn. Enzyme c s dng trong cc k thut sinh hc phn t nh RFLP, PCR, tch dng phn t......... Ni dung ca chng gm 3 vn c bn: (1). Enzyme gii hn, (2). cc nhm enzyme khc; (3). Enzyme nuctease. H thng enzyme c th chia thnh 6 nhm tu theo loi hnh phn ng m cc enzyme ny xc tc. Nhm enzyme nuclease c tc dng ct v ph hu ADN, nhm ADN-ligase c chc nng ni cc trnh t ADN li. nhm enzyme ADN-polimerase tham gia tng hp chui polinucleotit, nhm enzyme ADN-polimerase sa cha c chc nng sa cha nhng sai st trong phn t ADN, nhm enzyme topoisommerase tc dng m xon ADN, nhm enzyme phin m ngc reverse-transcriptase xc tc cho qu trnh phin m ngc t ARN thnh cADN. Cc nhm enzyme trn c vai tr ht sc quan trng trong cc thao tc vi ADN v trong cng ngh ADN ti t hp. Trong s c cc nhm enzyme gii hn, enzyme sa cha ADN, ADN-polimease, ADN-ligase....

1. ENYME GII HN (RESTRICTION ENZYME HOC ENZYME CT HN CH (RESTRICTION ENDONUCLEASE - RE) 1.1. Khi nim Nhm enzyme nuclease gm DN-ase v RN-ase c kh nng b gy lin kt phosphodiester kt qu l phn hu phn t ADN hoc ARN. Nhm enzyme ADN-ase gm 2 loi: Exonuclease (enzyme ct ADN ngoi bo) c tc dng ct tng cp nucleotit t hai u ca on ADN i dn vo bn trong (hnh 4.1). Enzyme exonuclease c th ct ta tng cp nucleotit c hai mch (ct kp) hoc ct tng nucleotit trn mt si ADN (ct n). Endonuclease (enzyme ct ADN ni bo) c tc dng ct ri ADN ti nhng vng xc nh gm vi cp nucleotit nm cnh nhau nh v trong phn t ADN, to thnh nhng phn on ADN nh hn. Enzyme endonuclease c th ct n

50

hoc ct kp (hnh 4.1).

Hnh 4.1. c im ct ca exonuclease v endonuclease

Mt loi enzyme endonuclease c th nhn bit v tr nht nh bn trong phn t ADN v ct c hiu, ct mt ln ti mt im . Nhng kt lun trn c pht hin trong thp k 60 ca th k XX. Trong nhng th nghim vi khun ngi ta pht hin mt nhm cc enzyme c th nhn bit v phn ct phn t ADN ca phag khi ADN ngoi lai ny bo v t bo ch xm nhp. Cc enzyme ny bo v t bo ch khi b nhim phag - l cc enzyme gii hn. Enzyme ct hn ch (enzyme gii hn) l loi enzyme nuclease c kh nng nhn bit c im ct v ct ti im xc nh. Tuy nhin vn t ra l, liu enzyme gii hn ct ADN ca chnh t bo ch? Nhng nghin cu cho thy cc enzyme gii hn phn hu bt k ADN ngoi lai no xm nhp vo t bo; trong t bo h thng enzyme sa i (methylase) sa i ADN t bo ch bng cch methyl ho cc baz xc nh ca on trnh t nhn bit, nh vy ngn cn enzyme gii hn ct vo ADN ca t bo ch. Nhng enzyme ny l i din ca mt trong cc nhm enzyme quan trng nht 51

dng trong thao tc ADN. 1.2. Cc nhm enzyme gii hn Cc enzyme gii hn c ba nhm I, II, III v cc enzyme gii hn c dng ph bin ngy nay thuc nhm II. Enzyme gii hn nhm II c c ch tc ng n gin nht, chng l cc nuclease, v v chng ct ti mt v tr nm bn trong si ADN v khng phn hu ADN t hai u, nn gi l endonuclease. Bng 4.1. c im ca cc loi enzyme gii hn

c im Nhm I Nhm II Nhm III im ct Xa im nhn bit

hn 1000 bp Nm trong im nhn bit

Nm ngoi im nhn bit nhn bit

Kh nng methy ha gc Adenine

C Khng C

iu kin ct ATP,Mg++, Mg++ S-AdoMet

Mg++ hoc Mn++ Mg++, S-AdoMet

Cu trc ca enzyme (s chui polipeptide)

Khc nhau Ging nhau Khc nhau

Hnh 4.2. Qu trnh methyl ho bi enzyme methylase

Cch gi tn cc enzyme gii hn da trn cc quy c quc t. Tn chi v tn loi ca sinh vt, m tm thy enzyme, c dng t cho phn

52

u ca tn enzyme, phn gm ch th nht ca tn chi v hai ch u ca tn loi. Chng hn enzyme tch chit c t mt ni ca Escherichia coli th t tn l Eco, cn enzyme tch t Bacillus amiloliquefaciens th gi l Bam... Hai loi enzyme c s dng rng ri c tch ra t cc vi khun ni trn l EcoRI v BamHI. V d: EcoRI: RE tch t vi khun E. coli, dng R, nhm I. HindIII: RE tch t vi khun Heamophilus influenzae, dng d, nhm III. Enzyme gii hn c tnh c hiu, mi enzyme c th nhn bit mt on trnh t c th cc baz trn ADN, on nhn bit ph bin nht c chiu di t 4 - 6 bp. Nu mt trnh t ADN c 4 bp th c 44 = 256 bp lp li mt ln, trnh t c 5 bp th c 45 = 1024 bp lp li, trnh t c 6 bp th c 46 = 4096 bp mi lp li. Chng hn, mt enzyme nhn bit trnh t c 4 bp s to ra on ngn hn so vi trnh t c 6 bp. Loi hnh ca cc on ADN do mt enzyme c th to ra tu thuc vo trnh t nhn bit v vo v tr ca im ct trong trnh t ny. Nh chng ta thy, chiu di ca on ny ph thuc vo tn s lp li ca trnh t nhn bit. im ct thc t ca enzyme quyt nh hnh dng ca u b ct. Hnh dng ny rt quan trng i vi vic thao tc tip ADN. C hai kiu hnh dng cc on c th sinh ra: (1) Cc on u bng (blunt ends); (2) Cc on u dnh (so le) vi 2 kiu: on c u 3' nh ra; v on c u 5' nh ra.

Hnh 4.3. Cc kiu ct ca RE nhm II

Cc enzyme nh PstI v EcoRI to ra cc on ADN c u dnh, chng c th bt cp baz vi trnh t b sung cng do chnh enzyme to ra. Nh vy, bng cch ct hai mu ADN khc nhau bi cng mt enzyme v trn cc 53

on vi nhau c th to ra ADN ti t hp. Bng cch ny cc on ADN ngun khc nhau c th ni li vi nhau nu chng c cc u dnh do enzyme gii hn sinh ra. Khi cc vng b tr ghp vi nhau th b khung photphodiester c gn li bng ADN ligase. Trong thc nghim, ngi ta trn mt lng thch hp enzyme vi ADN caanf nghin cu trong dung dch m, cho phn ng din ra 370C. Hot tnh enzymc c do bng n v tng ng vi lng enzyme cn thit mt microgam ADN trong mt gi 370C. 1.3. Lp bn ct gii hn Phn ln cc on ADN u c cc im nhn bit i vi nhiu enzyme gii hn khc nhauv nu bit c v tr tng i ca mt s im ny th c th lp c bn cc im gii hn. Trong k thut ny, ngi ta s dng cc enzyme gii hn ct mt on ADN, sau in di trn agrose, chp nh phn tch c kch thc cc on ADN b ct. T cc kt qu phn tch c th xc nh c v tr tng i ca cc im c ct trn phn t ADN.

Hnh 4.4. Kiu ct ca mt s loi enzyme

Gi s chng ta mun lp bn cc im ct ca cc enzyme BamHI, EcoRI v Psd i vi on ADN c chiu di 15kb, kt qu thu c nh sau: Nu ch s dng ring l mt loi enzyme th on ADN di 15 kb s b ct thnh 2 on c kch thc khc nhau. BamHI ct on ADN thnh 2 on vi kch thc 14 kb v 1 kb EcoRI ct on ADN thnh 2 on vi kch thc 12 kb v 3 kb PstI ct on ADN thnh 2 on vi kch thc 8 kb v 7 kb

54

Nu s dng phi hp 2 hoc 3 loi enzyme th on ADN b ct thnh 3 hoc 4 on nh: BamHI + EcoRI ct on ADN thnh 3 on vi kch thc 11 kb, 3 kb v 1 kb. BamHI + PstI ct on ADN thnh 3 on vi kch thc 8 kb, 6 kb v 1 kb. EcoRI + PstI ct on ADN thnh 3 on vi kch thc 7 kb, 5 kb v 3 kb. BamHI + EcoRI+PstI ct on ADN thnh 4 on vi kch thc 6kb, 5kb, 3kb, 1kb.

Hnh 4.5. Cc enzyme BamHI, EcoRI v PstI ct trnh t 15kb thnh 2 phn

on ADN c kch thc khc nhau Vic pht hin cc enzyme gii hn l bc t ph quan trng trong lnh vc sinh hc phn t ni chung cng nh cng ngh ti t hp AND ni ring. Thc cht nhng enzyme dng trong cng ngh ADN ti t hp thuc enzyme gii hn nhm II. Chng phn ct khu vc bn trong phn t ADN ti mt trnh t nucleotit nht nh v to ra hn hp (qun th) cc on ADN vi kch thc khc nhau. Cho n ngy nay ngi ta tch c hn 1200 enzyme gii hn nhm II t cc c th procariot. Trn 70 enzyme gii hn hin ang c bn sn trn th trng. Cc RE c th nhn bit nhng trnh t ngn ca ADN v ct ng vo v tr phn chia phn t ADN thnh nhng on c di c trng. Cc RE khc nhau th c on nhn bit vi trnh t baz c trng khc nhau. S m t cc v tr ct khc nhau ca mt phn t ADN c gi l bn ct hn ch. Nhim sc th ca Eukaryot cha phn t ADN gm nhiu gene v n c th b ct bi enzyme ct hn ch thnh nhiu on nh, chnh v vy c th s dng enzyme gii hn trong ng dng lp bn ct hn ch.

55

Hnh 4.8. in di cc phn on ADN b ct bi s phi hp cc enzyme

a. BamHI + EcoRI b. BamHI + PstI c EcoRI + PstI d. BamHI + EcoRI+PstI Quy trnh lp bn ct hn ch theo quy trnh di y:

1. Nghin cu a dng kiu hnh trong qun th. 2. Tch chit phn t ADN. 3. S dng enzyme RE. 4. in di trn gene agarose.

Phn on ct bi RE-A ADN chun Phn on ct bi RE-B

Hnh 4.7. S cc bng in di ca cc mu th nghim v ADN chun Ngoi ng dng thit lp bn ct gii hn ngi ta cn s dng enzyme gii hn kt hp vi enzyme ni to vector ti t hp (hnh 4.8).

56

Hnh 4.8. S dng enzyme gii hn v enzyme ni to ADN ti t hp

2. CC NHM ENZYME KHC 2.1. Polimerase Cc polimerase tham gia vo qu trnh ti bn ADN v phin m tng hp ARN v c s dng nhiu trong k thut di truyn. Cc pohmeasase gm 2 nhm: (1) ADN-polimerase xc tc tng hp chui polinucleotit theo chiu t 5' n 3'. Taq polimerase (enzyme tch t vi khun sui nc nng) c kh nng chu nhit, s dng trong phn ng chui PCR. Enzyme phin m ngc (Reverse Transcriptase: RT-ase) xc tc tng hp CADN t khun ARN; (2) Cc ARN-polimerase tham gia tng hp ARN. Ngoi cc enzyme trn cn c nhiu enzyme tham gia sa cha nhng sai st trn phn t ADN. 2.1.1. Taq polimerase Taq polimerase c tch chit t mt loi vi khun Thermus aquaticus sui nc nng. Taq polimerase c s dng trong PCR v RAPD tng hp on ADN trong iu kin c dung dch m, Mg+ primers. Hin nay cc nh khoa hc c th phn lp gene xc nh enzyme polimerase, tch dng v biu hin gene ny. Chnh v vy bng cng ngh ADN ti t hp c th to ra lng ln enzyme Taq polimerase s dng trong k thut di truyn. 2.1.2. ADN-polimerase I Enzyme ADN-polimerase I, ngoi chc nng polimerase, cn c hot tnh

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ca exonuclease 5' 3' v 3' 5'. Enzyme ny xc tc phn ng thay th si, trong phn ng chc nng exonuclease 5' 3' phn hu si khng lm khun trong khi polimerase tng hp bn sao mi. Chc nng exonuclease 5'3' ca ADN polimerase I c th c loi b bng cch ct enzyme to ra on Klenow. on ny vn duy tr cc hot tnh polimerase v exonuclease 3' 5'. on Klenow c s dng khi cn sao chp mt phn t ADN mch n. 2.1.3. Enzyme phin m ngc Enzyme phin m ngc c pht hin t 1970 c chc nng tng hp ADN t ARN. Trong t nhin, enzyme ny do Retrovirus sn sinh phin m ngc t h gene ARN ca chng thc hin qu trnh xm nhp vo h gene ca t bo ch. Reverse Transcriptase c s dng trong k thut to cADN bng phn ng PCR ngc (RT-PCR). Hin nay ngi ta s dng cc loi enzyme phin m ngc nh: Avian Myeloblastosis virus reverse transcriptase (AMV-RT) v Moloney Murine Leukemia virus reverse transcriptase (M-MLV-RT), trc y AMV-RT phi tch chit t virus Myeloblastosis gia cm, cn M-MLV-RT t virus gy bnh Leukemia chut. Cc enzyme ny l sn phm ca gene pol ca virus v hin nay cc enzyme ny nhn c t cng ngh ADN ti t hp. Enzyme phin m ngc l mt ADN-polimerase ph thuc ARN, v vy n sn sinh ra si ADN t si khun ARN. N khng c hot tnh exonuclease km theo. Enzyme ny ch yu c s dng sao chp cc phn t mARN khi chun b cADN (ADN b tr hoc ADN bn sao) tch dng, mc d n cng c tc ng trn khun ADN. 2.1.4. T4 ADN-polimerase Enzyme T4 ADN-polimerase c ngun gc t phage T4 k sinh trong t bo vi khun. Hot tnh ca enzyme ny ging nh hot tnh on Klenow v c s dng trong tng hp mu d c phng x cao. Bi v hot tnh exonuclease 3'-5' mnh hn T4 ADN-polimerase. 2.1.5. Terminal transferase Enzyme Terminal transferase c li trch t tuyn c b, c chc nng xc tc phn ng gn cc deoxynucleotit vo u 3'-OH t do ca ADN. Terminal transferase c s dng khi thm ui polinucleotit to u so le cho ADN trong k thut to dng. 2.1.6. Enzyme Alkaline phosphatase Enzyme Alkaline phosphatase c phn lp t E. coli hay t rut b. Chc nng ca enzyme ny l loi b nhm 5' phossphate ca ADN, ARN v

58

cc nucleotit t do. Enzyme Alkaline phosphatase s dng loi b 5'phossphate ca ADN v ARN trc kho nh du phng x bng 32P; s dng trong k thut to dng, khi vector c "m" bi RE v enzyme lm mt 5' phosphate vector khng th ni li c. 2.1.7. Enzyme T4 polinucleotit kinase Enzyme T4 polinucleotit kinase c ngun gc t phagene T4 k sinh trong E. coli. Chc nng ca enzyme T4 polinucleotit kinase ngc li vi Alkaline phosphatase, xc tc cho phn ng thm vo nhm phosphate u 5' ca ADN hay ARN. T4 polinucleotit kinase s dng nh du phng x u 5' ca ADN trong k thut xc nh trnh t ADN v phosphoryl ho cc trnh t ADN khng c nhm phosphate u 5' chun b cho phn ng ni ca k thut to dng. 2.2. Enzyme ni ADN-ligase Enzyme ni ADN-ligase xc tc s hnh thnh lin kt ni hai on ADN v ARN. ADN-ligase l mt enzyme ni quan trng trong t bo v chc nng ca n l sa cha cc mi lin kt photphodiester b t gy mt cch ngu nhin hoc trong ti bn ADN hay ti t hp ADN. ADN-ligase c s dng trong k thut to dng v thng c kt hp vi cc enzyme Alkaline phosphatase v polinucleotit kinase. Nhm enzyme ligase c s dng nhiu nht trong cc th nghim vi ADN v ARN ch yu gm E. coli ADN-ligase, T4 ADN-ligase v T4 ARN-ligase. 2.2.1. E. coli ADN-1igase Enzyme E. coli ADN-ligase c tch chit t E. coli, xc tc cho cc phn ng ni hai trnh t ADN c u dnh (so le).

Hnh 4.9. Enzyme E. coli xc tc phn ng ni hai trnh t c u dnh 2.2.2. T4 ADN - ligase Enzyme T4 ADN-ligase chit t phage T4 k sinh E. coli, xc tc cho phn ng ni hai trnh t c u bng, c s dng nhiu trong k thut to 59

dng. 2.2.3. T4 ARN-ligase Enzyme T4 ARN-ligase chit t phage T4 k sinh E. coli, xc tc cho phn ng ni hai trnh t ARN bng lin kt phosphodiester. Enzyme T4 ARN-ligase thng c s dng nh du phng x u 3' ca ARN trong k thut sn xut mu d (probe). Nh vy, enzyme ligase c hiu qu trong s gn cc khe h ca cc trnh t c u dnh hoc u bng vi nhau trong nhng iu kin thch hp. Nhit thch hp l 370C, nhng n c th hot ng nhit thp hn nhiu (4 15 0C).

3. ENZYME NUCLEASE Enzyme nuclease l nhm enzyme phn ct ADN (ADNse) v ARN (ARNse) 3.1. DN-ase I Enzyme DN-ase I c kh nng ct lin kt ngay sau mt baz pyrimidine trn ADN mch n hay mch kp. Trong thc nghim DN-ase I c s dng loi ADN trong ch phm ARN v protein, to im t trong k thut nh du mu d. 3.2. Nuclease S1 Enzyme nuclease S1 tch chit t Aspergillus oryzae c tc dng phn gii ADN mch n, mch kp. Enzyme nuclease S1 c s dng phn tch c im cu trc phn t lai ADN - ARN, loi b u so le to u bng, loi b cc nt vng trn ARN trong phn ng phin m ngc to cADN. 3.3. Exonuclease III Ezyme Exonuclease III chit t E. coli xc tc phn ng phn gii tun t cc nucleotit t u 3'- OH t do ca ADN theo chiu t 3'5' to ra nhng vng mch n trn ADN mch kp. Enzyme Exonuclease III c ng dng to cu trc mch n ca mt vng trn phn t ADN, to t bin mt on trn ADN khi phi hp vi nuclease S 3.4. RN-ase A Enzyme RN-ase A c hot tnh rt mnh, c th chu ng c nhit 900C trong 60 pht. Enzyme RN-ase A c kh nng ct lin kt phosphodiester ngay sau mt baz pyrimidine ca ARN. Enzyme RN-ase A c s dng loi b ARN trong ch phm ADN hoc protein, loi b cc on khng bt cp

60

trn ARN ca phn t li ARN -ADN. 3.5. RN-ase H Enzyme RN-ase H c kh nng loi b ARN trong phn t lai ARN - ADN v thng c s dng loi ARN sau phn ng phin m ngc tip tc tng hp mch th hai ca CANAto ADN mch kp. THO LUN

1. Th no l enzyme ct v enzyme ct hn ch? Cc nhm enzyme gii hn.

2. Vn c im ca cc loi enzyme dng trong k thut sinh hc phn t.

3. Trnh by cc kiu ct ca enzyme gii hn, cho v d. Nu nhng ng dng c bn ca RE.

4. Th no l bn ct gii hn? cho v d minh ho. 5. Nhm enzyme nhn ADN v ni ADN. 6. Nghin cu ch th sinh hc phn t bng phng php s dng cc loi

enzyme ct hn ch.

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Chng 5: XC NH MI LIN QUAN GIA PROTEIN V C TNH SINH VT Tm tt: Protein l sn phm biu hin gene, protein biu hin thnh tnh trng v c tnh sinh vt. Trong t bo c nhiu loi protein chc nng quan trng v nhng hiu bit v c im ca nhng protein ny l c s cho nhng ng dng vo cng tc chn ging v bo v sc kho con ngi. Nhng loi protein cp trong chng ny l protein lin quan n tnh chng chu ca sinh vt v protein c ch hot ng ribosome. Xc nh mt lin quan gia protein v tnh trng, c tnh sinh vt lp thnh cc ch th protein cho c tnh v tnh trng. Cc ch th protein c th l ch th hm lng protein, enzyme, phn t protein hay ph isozyme v xc nh cc loi ch th ny c thc hin theo quy trnh k thut nht nh. Hm lng protem, ng v hot enzyme lin quan n p sut thm thu trong t bo v quan h vi tnh chng chu ca sinh vt. C nhiu c tnh v tnh trng ca sinh vt c quan tm nghin cu hin nay l tnh chng chu ca sinh vt v vic tm ra cc ch th protein cho c tnh ny c ngha cao trong chn to ging. Ni dung ca chng gm 6 vn c bn: (1). Mt s loi protein chc nng; (2). Quy trnh xc nh ch th protein; (3). K thut phn tch protein; (4). K thut phn tch enzyme; (5). K thut phn tch ng, (6). Nghin cu RIPs bng Westem blot.

1. MT S LOI PROTEIN CHC NNG 1.1. Protein lin quan n tnh chng chu Kh nng chng chu ca sinh vt ni chung v ca thc vt ni ring lin quan n cu trc v chc nng ca cc thnh phn trong t bo. Cc vng t bo v cc qu trnh xy ra trong mi vng lin quan n kh nng chu hn, nng, mn, phn ca thc vt (hnh 5.1).

62

Hnh 5.1. Chc nng ca cc vng t bo khi mi trng thiu nc (theo

Bohnert,Jensen, 1996 v inh Th Phng, 2001)

Hnh 5.2. Hot ng ca cc gene lin quan n s mt nc v s thay i p

sut thm thu (theo Bray, 1993 v Trn Th Phng Lin, 1999). 1.1.1. Protein sc nhit (HSP) HSP c hu ht cc loi thc vt nh: la m, mch, la go, ng, u hnh, ti.... chng chim khong 1% protein tng s trong l ca cc loi thc vt ny. HSP cn tm thy vi khun v ng vt bc cao. HSP c tng hp khi t bo gp iu kin cc oan nh hn, nhit cao, mui cao.

63

HSP c th chia lm 6 nhm da trn c s khi lng phn t khc nhau: MW 110, 90, 70, 60, 20, 8,5 kDa. Trong s c nhm HSP70 v HSP60 c nhiu i din ca cht mi gii phn t (gi l chaperonin). HSP8,5 hoa (ubiquytin) c chc nng bo v cho t bo nhng khng phi l mi gii phn t. Ubiquytin c hot tnh protease vi chc nng phn gii cc protein khng c hot tnh enzyme, ngn chn hin tng gy c t bo ca nhng protein ny. Ubiquytin c MW rt thp, t chu nh hng ca nhit nn ubiquytin c vai tr t sa cha ca t bo khi gp yu t cc oan, c bit l nhit . 1.1.2. Mi gii phn t (MGPT) Theo Ellis (1997) MGPT l mt nhm gm nhiu loi protein khc nhau, nhng chng u c chc nng tham gia to cu trc khng gian ng cho protein trong t bo. Chc nng ca MGPT th hin nhng ni dung sau: - Gi n nh chui polipeptit khi ang tng hp v to cu trc khng gian ng cho chng. - To li cu trc khng gian ca protein sau khi vn chuyn qua mng t bo. - Lp gip cc chui polipeptit vo phc to cu trc bc 4. - MGPT c tng cng nh qu trnh tng hp trong iu kin cc oan do nhu cu cp thit ca t bo. Hot ng ca MGPT c thc hin nh vic s dng nng lng ATP. MGPT c nghin cu u tin thc vt v lin quan n enzyme Ribulose bisphotphat cacboxylase/oxygenelase (RUBISCO). RUBISCO xc tc cc phn ng tng hp cht hu c nh s dng CO2 t do trong quang hp. RUBISCO c cu trc bc 4 gm t 4- 8 tiu n v ln (MW52) v 8 tiu n v nh (MW14 kDa). Mt loi protein c hiu gn vi nhng tiu n v chnh to phc RUBISCO-binding protein (RBP) v RBP l MGPT. MGPT thc vt tham gia to cu trc khng gian ng cho nhng phn t protein mi c tng hp, chuyn protein qua mng, duy tr cu trc c hiu ca protein, ngn chn s hu hoi protein cha to cu trc ng, khi u s phn gii protein b bin tnh. MGPT c th phn thnh mt s nhm ch yu sau:

HSP70 l nhm MGPT c hot tnh ATP-ase v c tnh bo th cao. Trong iu kin cc oan HSP70 c th ngn chn s co cm v kt t protein.

HSP60 (chaporonin) l loi protein cu to t cc tiu n v c khi lng 60 kDa v cng c hot tnh ATP-ase. Chng c cu to 2 vng,

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mi vng c t 7-8 tiu n v. t bo Prokaryot, chaperonin ging vi protein tm thy ti th, lc lp v c gi l GroEL, GroES. Trong t bo Eukaryot tm thy mt loi chaperonin trong t bo cht l CCT. Nghin cu v CCT thc vt cn t c cp. Tuy nhin gn y c nhng cng b u tin ca Nng Vn Hi v cng s (1998), Trn Th Phng Lin v cng s (1999) v kt qu phn lp v tch c dng gene CCT t cy u tng.

1.1.3. HSP100, HSP90, SHSP Cc nhm HSP100, HSP90, sHSP c tnh bo th cao v c hot tnh ATP-ase. Phn ln cc protein ny c sinh ra khi.gp nhit cao. Chc nng ca HSP100, HSP90, SHSP l ngn chn hin tng co cm ca protein v ti hot ho cc phn t protein bin tnh. 1.1.4. LEA (Late embryogenesis abundant protein) LEA l loi protein c tng hp vi s lng ln trong giai on cui ca qu trnh hnh thnh phi. LEA c vai tr quan trng i vi kh nng chu kh hn ca ht. Cu trc phn t ca LEA ni bt l giu axit amin a nc, khng cha xystein, tryptophan, c vng xon v c kh nng chu nhit. Chng thc hin cc chc nng c lp ion, bo v mng, hu protein bin tnh v iu chnh p sut thm thu. LEA c chia lm 6 nhm: nhm 1-D19; nhm 2-D11 (dehydrin); nhm 3-D7; nhm 4- D113; nhm 5-D29; nhm 6-D95. Nhiu gene LEA c nghin cu, phn lp v xc nh chc nng. Khi gp hin tng mt nc mARN ca n c tng hp v dn tr thnh mt lng ln gm nhiu loi mARN khc nhau trong ht chn, mARN b phn gii ht khi ht ny mm. Mc phin m ca gene LEA c iu khin bi ABA v mt nc ca t bo. 1.2. Protein bt hot ribosome (Ribosome inactivating proteins - RIPs) Protein bt hot ribosome (ribosome inactivating proteins - RIPs) l nhng c t (toxin) c hot tnh rRNA N-glicosidase v cc c tnh sinh - dc hc rt a dng. C ch tc ng ca RIPs l phn ct cu ni N- glicosidic ca adenine c hiu trn vng Sarcin/Ricin Loop (cn c gi l SRL hay R/S Domain) ca rRNA (v tr A4324 trn rRNA 28S gan chut). Kt qu l ribosome khng gn kt c vi EF-2 v GTP, lm c ch qu trnh sinh tng hp protein. RIPs c phn loi theo cu trc phn t ca chng theo hai hoc ba tipe khc nhau: (1) RIPs tipe I ch c mt chui polipeptit A (i khi dng glicosyl ho) vi hot tnh RIPs N-glicosidase; (2) RIPs tipe II c hai chui: polipeptit A lin kt vi chui plipeptit B (l mt phn t c hot tnh ging lectin) bng mt

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hoc hn cc cu ni disulfide; (3) RIPs tipe III ging tipe I, bao gm mt chui polipeptit, nhng li ch c hot tnh sau khi c phn ct on tn hiu bng mt loi protease c hiu. S lng RIPs tipe I (khi lng phn t khong 30kDa) nhiu hn RIPs tipe II v III. Tuy nhin mt s RIPs khng th phn loi theo cc tipe k trn. RIPs c pht hin hn 50 loi thuc 13 h thc vt, bao gm c cy mt v hai l mm. RIPs khng ch thy h Bu b (Cucurbitaceae) vi rt nhiu loi l cc cy thuc qu nh Trichosanthes, Luffa, m cn thy rt nhiu cc h thc vt khc nh Cm chng (Caryophylaceae), Thu du (Euphorbiaceae), Ho tho (Poaceae), H u (Fabaceae), h Hoa phn (Nygtaginaceae)... RIPs khng ch tm thy thc vt, m cn thy mt s loi vi khun ng rut. S c mt ca RIPs rt nhiu c th sinh vt khc nhau a n mt gi nh v kh nng tn ti ca loi protein tng t th gii ng vt.

2. QUY TRNH XC NH CH TH PHN T PROTEIN Protein l sn phm ca s biu hin gene. Protein biu hin tnh trng ca c th. Nghin cu xc nh c s protein ca tnh trng hnh thi, cu to, sinh l, ho sinh ca c th sinh vt c quan tm mt s kha cnh c th nh hm lng protein, cc tiu phn protein vi kch thc phn t khc nhau. Quy trnh xc nh ch th protein cho mt tnh trng no c th c thc hin theo cc bc sau: Bc 1. Nghin cu c tnh hnh thi, sinh l Sng lc, phn lp cc c th theo kt qu nh gi da trn kiu hnh. Nhng c th mang tnh trng quan tm c th c to ra do lai ging hoc t bin thc nghim, sng lc trong qun th t nhin v cng c th do x l, nh gi trong iu kin nhn to. Hin nay ngi ta quan tm ch yu cc tnh trng chung chu thc vt (kh nng chu nhit: nng, lnh; mn; hn; phn; kh nng chu su, khng bnh), cc bnh do vim hoc ung th ng vt. Bc 2. Xc nh ch th protein

Ch th hm lng protein - Chit protein giai on sinh trng, pht trin quan tm ca c th. - Phn tch hm lng protein. - Xc nh mi lin quan n tnh trng.

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Ch th phn t protein - Chit protein dng cho in di, tch chit phn on protein. - Phn tch thnh phn in di. - Phn tch a hnh protein. - Gii trnh t axit amin ca protein.

Western blot - Chit protein dng cho in di. - Tch protein bng in di trn gel poliacrylamide. - Chuyn protein t gel sang mng (PVDF) nh in trng.

Ch th enzyme - Chit enzyme.

- Phn tch nh lng v nh tnh hot enzyme. - Phn tch in di entyme.

Hnh 5.3. S phn tch xc nh ch th protein

3. K THUT PHN TCH PROTEIN 3.1. Tch chit protein Protein c tnh tan trong nc v mui, do vy ngi ta chit protein bng mt s loi dch chit c pH khc nhau kt hp vi phng php li tm thu

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dch chit. Sau y trnh by cch chit protein s dng cho nh lng v in di protein. 3.1.1. Tch chit protein d tr trong ht thc vt Protein d tr trong ht c th c chit bng cc loi dch chit nh nc ct, NaCl, dung dch m phosphat xitrat pH = 10... i vi cc loi ht c du th trc khi chit protein cn phi loi lipit, v d k thut loi loi lipit ht u tng hoc u xanh: Ht u bc v, b phn phi mm sau nghin nh. Bt nhn c gi 40C. Loi lipit bng petrolium ete qua m 40C. Li tm 14.000 vng/pht loi dch li tm. Lp li mt vi ln na. Bt loi lipit gi 40C. 3.1.1.1. Chit protein dng phn tch nh lng hoc in di Bt loi lipit c s dng chit protein tng s bng dung dch NaCl 1M pH = 7.0, chit bng nc ct pH = 7.0 hoc chit bng m photphat pH=10. Bt loi lipit c chit bng dung dch m qua m 40C, trong 24 gi. Ngy hm sau li tm 14.000 vng/pht trong 30 pht thu dch pha trn. Dch li tm thu c cha protein tng s bao gm albumin v globulin. Trong nh lng protein c th tin hnh lp li nhiu ln; cn trong phn tch in di th c th thc hin mt ln. Dung dch protein c gi -200C. 3.1.1.2. Chit phn on protein cho phn tch in di Tch chit v phn on protein theo phng php Osborne v Klimenko. C th ly v d cy h u: Ht c nghin nh thnh bt mn, loi lipit bng ete petroleum sau tin hnh phn on. Protein c chit bng dung dch NaCl 1M v nc ct. Theo cch th nht, bt loi lipit c chit bng NaCl 1M sau li tm 14000 vng/pht trong 20 pht. Dch li tm cha albumin v globulin tng s (phn on I). Cn li tm cha tinh bt v tp cht c loi b. Tip theo albumin v globulin tng s c phn on bng cch thm tch, sau li tm. Dch li tm thu c cha phn ln albumin (phn on II). Cn li tm l cc protein khng tan trong nc m tan trong NaCl. Phn ny c ho vo NaCl 1M (phn on III). Theo cch th hai, protein c chit bng nc ct sau li tm. Dch li tm cha phn ln albumin v mt phn globulin (phn on IV).Cn li tm c chit tip bng NaCl 1M v li tm ly dch (phn on VII) Phn on IV c x l tip bng thm tch. Dch li tm cha phn ln albumin (phn on V). Cn li tm cha phn ln globulin tan trong NaCl 1M (phn on VI). 3.1.2. Tch chit protein trong m ng vt Chit protein t m ng vt n gin hn thc vt v c tin hnh

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tng t nh thc vt. Protein trong m ng vt c chit nh mui hoc nc v bng li tm. 3.2. nh lng protein 3.1.1. Phn tch hm lng nit tng s bng phng php Keldjal Nguyn tc : Tt c cc dng nit c trong c th hay trong cc m c gi l nh nit tng s. Nit c trong thnh phn axit amin ca protein l nit protein. Nit khng c trong thnh phn protein nh mui v c, axit nitric... l nit phi protein. Nit tng s = nit protein + nit phi protein. Oxi ho nguyn liu bng H2SO4 c mt cht xc tc v nhit cao, kt qu ca vic cng ph mu ny l m trong thnh phn cht hu c chuyn thnh (NH4)2SO4, di tc dng kim mnh, nng NaOH, sc NH3 bay ln gp nc to thnh NH4OH, NH4OH c H2SO4 th, Sau chun lng NH3 c H2SO4 bng NaOH. Kt qu phn tch s thu c hm lng ma nitg s bao gm c mi nittein v nit phi protein. Do vy cn tin hnh phn tch hm lng nit phi protein xc nh hm lng nit protein m chng ta quan tm trong nh gi cht lng sn phm sinh hc. Xc nh hm lng nit phi protein c th c xc nh theo phng php ca Barstein trn my phn tch m t ng Vapodest ca hng Gerhardt v my Kjeldahl. Hm lng nh pitotein = Hm lng nit tng s - Hm lng nitow phi protein 3.1.2. Phn tch hm lng protein tan tng s Nguyn tc chung l s dng cc thuc th nh Folin, Bradford, Gordan... vi s hp th nhng bc sng khc nhau trn my quang ph Uvis ng thi vi s phn tch mu th nghim l s dng protein chun bit trc hm lng tnh hm lng protein ca mu th nghim.

Phng php nh lng protein tan tng s theo phng php Lowry. C s ca phng php l s hnh thnh phc cht biur. Phc ny tham gia vi thuc kh Folin-ciocalteu cho mu c trng. Cng mu t l vi hm lng protein. o cng mu ca hn hp trong ng nghim trn my so mu quang in bc sng = 750nm. Sau khi o c mt quang th em i chiu vi th chun tm lng protein tng ng. Th ngh